Synthesis of Mitochondria-Anchored Nitroimidazoles with a Versatile NIR Fluorophore for Hypoxic Tumor-Targeting Imaging and Chemoradiotherapy

Article abstract of DOI:10.1021/acs.jmedchem.0c02250

Nitroimidazoles are one of the most common radiosensitizers investigated to combat hypoxia-induced resistance to cancer radiotherapy. However, due to poor selectivity distinguishing cancer cells from normal cells, effective doses of radiosensitization are much closer to the doses of toxicity induced by nitroimidazoles, limiting their clinical application. In this work, a tumor-targeting near-infrared (NIR) cyanine dye (IR-808) was utilized as a targeting ligand and an NIR fluorophore tracer to chemically conjugate with different structures of hypoxia-affinic nitroimidazoles. One of the NIR fluorophore-conjugated nitroimidazoles (808-NM2) was identified to preferentially accumulate in hypoxic tumor cells, sensitively outline the tumor contour, and effectively inhibit tumor growth synergistically by chemotherapy and radiotherapy. More importantly, nitroimidazoles were successfully taken into cancer cell mitochondria via 808-NM2 conjugate to exert the synergistic effect of chemoradiotherapy. Regarding the important roles of mitochondria on cancer cell survival and metastasis under hypoxia, 808-NM2 may be hopeful to fight against hypoxic tumors.

Full text of DOI:10.1021/acs.jmedchem.0c02250

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Article
Synthesis of Mitochondria-Anchored Nitroimidazoles with a
Versatile NIR Fluorophore for Hypoxic Tumor-Targeting Imaging
and Chemoradiotherapy
Sha Chen,⊥ Songtao Yu,⊥ Zaizhi Du, Xie Huang, Meng He, Shuang Long, Jing Liu, Yu Lan, Dong Yang,
Hao Wang, Shuhui Li, An Chen, Yuhui Hao, Yongping Su, Changning Wang,* and Shenglin Luo*
Cite This: J. Med. Chem. 2021, 64, 3381−3391
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ABSTRACT: Nitroimidazoles are one of the most common radio-
sensitizers investigated to combat hypoxia-induced resistance to cancer
radiotherapy. However, due to poor selectivity distinguishing cancer
cells from normal cells, effective doses of radiosensitization are much
closer to the doses of toxicity induced by nitroimidazoles, limiting their
clinical application. In this work, a tumor-targeting near-infrared (NIR)
cyanine dye (IR-808) was utilized as a targeting ligand and an NIR
fluorophore tracer to chemically conjugate with different structures of
hypoxia-affinic nitroimidazoles. One of the NIR fluorophore-
conjugated nitroimidazoles (808-NM2) was identified to preferentially
accumulate in hypoxic tumor cells, sensitively outline the tumor
contour, and effectively inhibit tumor growth synergistically by
chemotherapy and radiotherapy. More importantly, nitroimidazoles
were successfully taken into cancer cell mitochondria via 808-NM2 conjugate to exert the synergistic effect of chemoradiotherapy.
Regarding the important roles of mitochondria on cancer cell survival and metastasis under hypoxia, 808-NM2 may be hopeful to
fight against hypoxic tumors.
delivery, and imaging-guided treatment. Into the tumor-targeted
parent structure of IR-808, some radionuclides,14,15 anticancer
drugs,16,17 and photosensitizers18,19 were introduced and
successfully used for tumor-specific multimodal imaging (NIR,
positron emission tomography (PET), or single-photon
emission computed tomography (SPECT)) and treatment
(chemotherapy, photodynamic, or photothermal therapy).
Although some conjugates exhibited excellent antitumor effects
in both in vitro and in vivo studies, their treatment toward
hypoxic tumors has not been tested. Hypoxic tumors are known
to be more resistant to chemotherapy and radiotherapy, leading
to poor prognosis and higher patient mortality.1−3 The presence
of hypoxic regions within tumors hinders the effect of both
chemotherapy and radiotherapy.20
INTRODUCTION
■
Hypoxia is a cardinal component of the tumor microenviron-
ment and plays a key role in tumor cell resistance to therapy,
disease progression, and metastasis.1 In particular, radiotherapy,
as one of the main strategies for cancer treatment, is sensitively
affected by hypoxia.2 To combat the hypoxia-induced resistance
to radiotherapy, several approaches have been pursued,
including efforts to reoxygenate the tumor, radiosensitize
hypoxic cells, and develop hypoxia-activated prodrugs.3−7
A
notable progress in clinic toward hypoxia-targeting cancer
treatment was made by nitroimidazoles as oxygen-mimetic
radiosensitizers.8,9 In their structures, the electron-affinic nitro
group is designed to react with DNA radicals produced by
ionizing radiation in a manner similar to oxygen. The formation
of these adducts leads to DNA strand breaks and subsequent cell
death. However, due to the poor selectivity distinguishing cancer
cells from normal cells, the effective doses of radiosensitization
are much closer to those of the toxicity induced by nitro-
imidazoles, severely limiting their clinical application.10
In recent years, a series of structurally inherent tumor-
targeting heptamethine indocyanine dyes, such as IR-808,11 IR-
780,12 and IR-783,13 have been discovered. These dyes exhibit
near-infrared (NIR) fluorescence emission and preferential
accumulation in tumor cells without ligand conjugation,
providing potential applications for tumor diagnosis, drug
In this work, we designed and synthesized different covalent
conjugations by introducing nitroimidazoles to the N-alkyl side
chains of IR-808 dye based on previous structure−activity
Received: December 28, 2020
Published: March 10, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c02250
© 2021 American Chemical Society
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Scheme 1. Structures and Synthetic Route of Different 808-Nitroimidazole Conjugates
Figure 1. In vivo and in vitro tumor-preferential accumulation of 808-NM2. (a) In vivo and ex vivo NIR fluorescent imaging of subcutaneous 4T1 tumor
xenograft. The ex vivo imaging of dissected main organs (He: heart, Li: liver, Sp: spleen, Lu: lung, Ki: kidney, In: intestine, Mu: muscle, Tu: tumor) was
performed 24 h after the injection of 808-NM2. (b) Confocal fluorescence microscope showed that 808-NM2 preferentially accumulated into cancer
cells (4T1, MCF-7 cells) rather than normal cells (MCF-10A). (c) Intracellular localization of 808-NM2 (red) in three different kinds of cancer cells
with mitochondria-specific probe MitoTracker (green).
relationship studies for tumor targeting.21,22 Our results in this
work showed that one of the 808-nitroimidazole conjugates
(808-NM2) exhibited hypoxic cancer cell preferential uptake
and effective growth inhibition due to a synergistic effect of
chemotherapy and radiosensitization. Furthermore, the dual-
modal therapeutic conjugate was specifically located in cancer
cell mitochondria, which are the crucial cellular organelles for
promoting cancer cell survival and metastasis in the hypoxic
environment. Hence, the reported conjugate also showed to
inhibit cancer cell migration or metastasis through in vitro and in
vivo preliminary experiments. This new 808-nitroimidazole
conjugate may present a versatile small molecule to integrate
hypoxic cancer cell targeting, NIR imaging, chemoradiotherapy.
imidazole or 5-nitroimidazole as the starting material. Their
synthesis was not a straightforward conjugation between IR-808
and nitroimidazoles because there was no reactive chemical
group available to directly link two structures. The synthetic
routes and methods are also shown in detail in Figures S1 and
S2. The successful synthesis of these conjugates was
1
characterized by H nuclear magnetic resonance (NMR), 13C
NMR, and high-resolution mass spectrometry (Figures S3 and
S4). The purity of all final compounds was above 99% (Figure
S5). Their optical spectra were determined and demonstrated
both the maximal absorbance and emission in the NIR region
(700−900 nm) with a high extinction coefficient (Figure S6 and
Table S1).
With the NIR fluorescent property, the cancer-targeting
ability of the three conjugates was evaluated on 4T1-bearing
tumor xenografts (Figures 1a and S7). In vivo NIR imaging
revealed that 808-NM2 exhibited tumor-preferential accumu-
RESULTS AND DISCUSSION
■
As shown in Scheme 1, different 808-nitroimidazole conjugates
were designed and chemically synthesized using 2-nitro-
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Figure 2. Hypoxia-induced increase in cancer cell uptake of 808-NM2 and mechanism studies. (a) Comparison of cell uptake under normoxic or
hypoxic conditions (21, 5, 1% O2) by the confocal fluorescent microscope images of 4T1 and MCF-7 cancer cells. (b) Comparison of cell uptake under
normoxic or hypoxic conditions by flow cytometry and quantitative analysis (left three: 4T1; right three: MCF-7). (c) Mechanism studies of cellular
uptake in tumor cells by the interventions of 2-DG and BSP pretreatment (left: 4T1; right: MCF-7).
lation at 24 h after injection. Ex vivo NIR imaging of the
dissected organs and tumors further revealed that 808-NM2
preferentially accumulated in tumors. For in vitro tests, the
uptake of 808-NM2 by 4T1 and MCF-7 cancer cells was
investigated and compared with that of the normal epithelial cell
line MCF-10A. Both fluorescence confocal images and flow
cytometric results revealed preferential 808-NM2 accumulation
in cancer cells (Figure 1b). Subcellular localization of 808-NM2
in 4T1, MCF-7, and Ln229 cancer cells (Figure 1c) further
demonstrated its specific accumulation in mitochondria.
Fluorescence confocal microscopy images showed that the red
fluorescence of 808-NM2 overlapped with the green fluo-
rescence of a mitochondrial tracker (MitoTracker) in all cancer
cell lines, suggesting that the nitroimidazole moiety was
successfully taken into mitochondria. Mitochondria are vital
organelles for cell survival, providing energy production, and
inducing apoptosis and autophagy.23 In cancer, mitochondria
play key roles in tumor cell proliferation, invasion, and
metastasis.24 Hence, the mitochondria-anchored conjugate
may greatly enhance the radiosensitization effect of nitro-
imidazoles. The mechanism of mitochondria targeting may be
ascribed to the heptamethine core with lipophilic cationic
property in the structure of IR-808, according to previous
structure−activity relationship studies.18,21,22 Because 808-
NM2 was synthesized by introducing nitroimidazoles to the
N-alkyl side chains of IR-808, the conjugation didn’t change the
heptamethine core and lipophilic cationic property. Hence, 808-
NM2 remained to have the capacity of mitochondrial targeting
accumulation.
Then, we investigated the cellular uptake of 808-NM2 under
hypoxic conditions. After exposure to different hypoxic
conditions (5% O2, 1% O2), cell uptake of 808-NM2 in both
4T1 and MCF-7 cancer cells was found to notably increase,
compared to that under normoxic condition (21% O2) (Figures
2a and S8). Flow cytometric analysis also quantitively confirmed
the results in both cancer cell lines (Figure 2b). To further
investigate the factors affecting cellular uptake of 808-NM2,
cancer cells were treated with a glycolysis inhibitor 2-deoxy-D-
glucose (2-DG). Results showed that glycolysis inhibitor 2-DG
significantly decreased the cellular uptake of 808-NM2 (Figure
2c), suggesting that glycolysis may play an important role in the
808-NM2 uptake. Therefore, the possible mechanism of hypoxic
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Figure 3. Growth and migration inhibition on hypoxic 4T1 tumor cells. (a) Relative cell viability of 4T1 cells detected by MTT 72 h after incubation
with different concentrations of 808-NM2 under normoxic condition (21% O2). (b) Relative cell viability of 4T1 cells 72 h after incubation with low
concentrations of 808-NM2 under normoxic or hypoxic condition (21, 5% O2). (c) Relative cell viability of 4T1 cells 72 h after a combined treatment
with 808-NM2 and ionizing radiation under hypoxic condition (5% O2). The results were expressed as the mean SD **p < 0.01, n = 6, compared to
the close-left group, respectively. (d) Clonogenic survival assay of 4T1 cells after different treatments at different oxygen levels (21, 5, 1% O2). (e)
Wound healing assay of 4T1 cells under hypoxia (5% O2) before (0 h) or after different treatments (24 h).
cancer cell uptake of 808-NM2 may be associated with glycolysis
metabolic pathway, which is similar to the previous reports of
tumor-targeting IR-808.25,26 In addition, the cellular uptake of
808-NM2 was also remarkably inhibited when preincubated
with sulfobromophthalein (BSP), a competitive inhibitor of
organic anion transporting polypeptide (OATP) transporters.27
Because of the higher expression of OATP protein on cancer
cells (MCF-7) over normal human cells (MCF-10A), the
significant difference of 808-NM2 uptake between MCF-10A
and MCF-7 cells indicated that OATP transporters either
involved in one of the cancer-targeting mechanisms of 808-
NM2.11
The inherent antiproliferative activity of 808-NM2 was
initially evaluated in cancer cells without ionizing radiation.
MTT assays demonstrated that the cell viability of 4T1 cells
gradually decreased with increasing concentrations (Figure 3a).
For example, cell viability was reduced to 10% after 72 h
incubation with 2.5 μM 808-NM2. Its antiproliferative effect was
also confirmed in MCF-7, A549, and Ln229 cancer cells (Figure
S9), indicating potent anticancer activity on a broad spectrum of
cancer cells. Notably, its anticancer activity was dramatically
enhanced when cells were incubated under hypoxic conditions
(Figure 3b). This result is possibly induced by more efficient
cellular uptake of 808-NM2 under hypoxic conditions. To better
observe the effect of radiosensitization, low concentrations (0−
0.625 μM) of 808-NM2 were incubated for 6 h under hypoxia
followed by ionizing radiation. It could be easily found that cell
viability at each concentration was gradually reduced when
ionizing radiation was applied from 1 to 3 Gy (Figures 3c and
S10). Specifically, nearly no cell viability was found after co-
treatment with 0.6250 μM 808-NM2 plus 3 Gy radiation under
hypoxia (1% O2, Figure S11). The results revealed that a
remarkable anticancer effect was obtained by a combination of
chemotherapy and radiosensitizing effect. The number and
morphology of cancer cells after the treatment also visually
illustrated the synergistic effect of chemotherapy and radio-
therapy (Figure S12). A clonogenic survival assay was
performed, as it is known as the “gold standard” to assess the
radiosensitizing effect of a radiosensitizer.28,29 The photographs
shown in Figures 3d and S13 intuitively demonstrate that colony
formation was almost completely inhibited under hypoxia
compared with normoxia, suggesting that 808-NM2 could act as
a radiosensitizer with a good radiosensitizing effect. As
mentioned before, hypoxia plays an important role in cancer
cell metastasis. The inhibition of cell migration was evaluated by
wound healing assays with cancer cells. As shown in Figure 3e,
cell migration was obviously inhibited by the 808-NM2-treated
ionizing radiation group under hypoxia, which was much more
effective than that under normoxia (Figure S14). This tendency
was also verified in MCF-7 cancer cells (Figure S15).
For the anticancer mechanism study, the mitochondrial
membrane potential was detected by flow cytometry with the
mitochondria-targeted probe rhodamine 123.30 The average
fluorescence intensity of Rho123 decreased remarkably after
ionizing radiation with 0.625 μM 808-NM2 (Figure S16),
indicating that the mitochondrial membrane potential was
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Figure 4. Anticancer mechanism of 808-NM2 on hypoxia tumor cells. (a) Detection of ATP in 4T1 cells 72 h after different treatments. The results
were expressed as the mean SD, **p < 0.01, compared to normoxia control; ##p < 0.01, compared to hypoxia control, n = 3. (b) Apoptosis assay of
4T1 cells by flow cytometry 24 h after different treatments. (c) Expression of VEGFR2, p-mTOR, p-Akt, and p-4EBP1 in 4T1 cells 72 h after different
treatments. Normoxia and hypoxia indicate 21, 5% O2, respectively.
severely affected. Second, the production of ATP was detected
and found to be significantly reduced in the 808-NM2 radiation
group (Figure 4a). Third, ionizing radiation-induced radicals,
specifically reactive oxygen species (ROS), in mitochondria
were determined and revealed that much more ROS in
mitochondria were produced in the 808-NM2 ionizing radiation
group than in the control (Figure S17). These hyperactive ROS
may attack mitochondrial DNA (mtDNA) and perturb
mitochondrial functions for growth and migration.31 It is also
well known that high ROS generation and severe damage to the
mitochondrial membrane potential easily trigger the apoptosis
pathway of cell death.32 Indeed, both early and late apoptosis
were increased after the combined treatment under hypoxia
(Figures 4b and S18). Furthermore, we found that vascular
endothelial growth factor receptor 2 (VEGFR2) protein
expression decreased after treatment (Figure 4c). VEGFR2 is
a predominant inducer of both normal and pathophysiological
angiogenesis. It is activated by the hypoxic response elements
within the VEGF gene promoter.33 In addition, there was a sharp
decrease in the expression of p-mTOR, p-Akt, and p-4EBP1
(Figures 4c and S19), which are the key proteins in the PI3K/
AKT/mTOR signaling pathway regulating cell growth, cell cycle
progression, and angiogenesis.34 These findings may provide
possible mechanisms not only for tumor growth inhibition but
also for metastasis suppression induced by mitochondria-
targeted 808-NM2.
repeated three times of ionizing radiation with a total dose of 12
Gy). The tumor growth curves showed that tumor growth was
arrested in the 808-NM2-treated group and significantly
suppressed in the 808-NM2 irradiation group (Figure 5a).
The mean tumor weight in the 808-NM2 irradiation group was
much less than that in the 808-NM2 alone group (Figure 5b,c).
The number of tumor nodules on each lung was counted under a
microscope and obviously decreased in the mice from the 808-
NM2-treated radiation group (Figure 5d). H&E staining of the
lungs showed that a great number of tumor cells had migrated
into lung organs in the control and 808-treated groups but not in
the 808-NM2 radiation group (Figure 5e). Tumor tissues were
routinely processed and immunohistochemically stained for
CD31 and CD34, which are well known as markers for benign
and malignant vascular tumors.35 The results revealed that the
expression of both proteins was obviously decreased in the 808-
NM2-treated radiation group compared with the control group
(Figure S21). Therefore, both in vitro and in vivo results reveal
that 808-NM2 has a synergistic antiproliferative and radio-
sensitization effect on tumor growth inhibition.
Finally, to explore the potential for translational applications,
the in vivo antitumor efficacy of 808-NM2 was further evaluated
in a patient-derived xenograft (PDX) mouse model. Cancer
specimens derived from clinical patients were collected, and
patient diagnostic characteristics are shown in Table S2. 808-
NM2 was intravenously injected into mice in the PDX model
group and sham-operated group. In vivo NIR fluorescent
imaging of the anesthetized mice at 24 h postinjection showed
that an intensive fluorescent signal was observed at the tumor-
implanted site rather than at the sham-operated site (Figure 6a),
indicating that 808-NM2 maintained preferential tumor
accumulation in the PDX model. Then, 4 Gy ionizing radiation
was given to patient-derived xenografts and repeated three times
with a total dose of 12 Gy. Tumor growth was inhibited
significantly in the 808-NM2-treated nonradiation group based
The in vivo antitumor efficacy of 808-NM2 was initially
elucidated in an aggressive 4T1 subcutaneous tumor xenograft
mouse model. After the tumors reached 100 mm3, all mice were
randomly divided into three groups: the blank control group, the
808-NM2 group, and the 808-NM2 combined with the ionizing
radiation group. According to the in vivo NIR imaging results,
808-NM2 preferentially accumulated in tumors 24 h after
injection and 4 Gy ionizing radiation was given to tumors 24 h
after the injection of 808-NM2 via mouse tail veins (Figure S20,
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Figure 5. In vivo anticancer effect on metastatic 4T1 subcutaneous tumor models. (a) Tumor growth curves of 4T1 tumor xenograft mice in different
groups were measured during the observation period. Purple and orange arrows indicate the days of drug injection or ionizing radiation, respectively.
**p < 0.01, compared to control; #p < 0.05, compared to 808-NM2 group, n = 6. (b) Photos of tumors dissected from 4T1 tumor xenograft mice. (c)
Tumor weight in different groups. (d) Number of tumor nodules in mouse lungs from different groups was counted by a microscope. (e) H&E staining
of lung (dark blue spots) from different groups for pathological histology analysis. The group of 808-NM2+4 Gy means three 4 Gy doses of ionizing
radiation for a total dose of 12 Gy.
on the tumor growth curves and the mean tumor weight (Figure
6b,c, compared to the control group). The inhibition was further
enhanced in the 808-NM2 combined irradiation group
(compared to the 808-NM2 group). The body weight of mice
was monitored during treatment, and no significant differences
among groups were observed, suggesting good tolerance of all of
the treatments (Figure 6d). Mice were sacrificed, and the tumors
grown from cancer specimens were retrieved at the end of the
observation period. The tumor tissues were immunohistochemi-
cally stained for CD31 and CD34 (Figure S22). Both results
demonstrated that they underwent a dramatic decrease in the
808-NM2 radiation group. The tumor tissues were further
subjected to Ki67 (Figure 6e) and VEGFR2 immunochemical
staining (Figure S23). The results showed that the expression of
all of these genes decreased after treatment, indicating a
potential antimetastatic effect by inhibiting tumor angiogenesis.
by introducing hypoxia-affinic nitroimidazoles into IR-808 as a
novel tumor-targeting ligand. First, 808-NM2 retained the
property of preferential tumor accumulation mediated by OATP
transporters, which are overexpressed in a variety of cancer cells.
Second, this new 808-nitroimidazole conjugate with two
nitroimidazole groups exhibited a hypoxia-induced increase in
cancer cell uptake. Third, it could exert a synergistic anticancer
effect of chemotherapeutic and radiosensitizing activities,
possibly due to a hypoxia-induced increase in cell uptake by
cancer cells. More noteworthy, 808-NM2 was found to be
specifically retained in cancer cell mitochondria, disrupted
mitochondrial function and energy production and remarkably
decreased the adaptation of cancer cells to hypoxic environ-
ments to sensitize the anticancer effect of ionizing radiation.
This 808-nitroimidazole conjugate reported in this work, with
simultaneous hypoxia-enhanced cancer cell uptake and multi-
modal therapeutic activities by targeting mitochondria, may
present a useful strategy to combat hypoxia-induced cancer
therapy resistance.
CONCLUSIONS
■
In summary, a multifunctional NIR fluorophore was developed
for preferential accumulation in hypoxic cancer cells, sensitive
NIR fluorescence imaging, effective tumor growth, and
metastasis inhibition synergistically by chemoradiotherapy.
This versatile NIR fluorophore, namely, 808-NM2, was
synthesized and screened out from different covalent conjugates
EXPERIMENTAL SECTION
■
Chemicals and Instruments. Chemicals and solvents used for
synthesis were purchased from commercial suppliers (Sigma-Aldrich or
Acros) and directly used without further purification. All reactions were
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Figure 6. In vivo anticancer effect on patient-derived xenografts. (a) In vivo NIR imaging of mice from the sham-operated group and PDX model group
24 h after the injection of 808-NM2. (b) Tumor growth curves in the PDX mouse model in different groups during the observation period. Purple and
orange arrows indicate the days of drug injection or ionizing radiation, respectively. #p < 0.01, *p < 0.05, compared to control, n = 3. (c) Tumor weight
in different groups. (d) Body weight of mice from different groups after treatment. (e) Immunohistochemical staining of Ki67 (dark brown spots) in
tumor sections from different treatment groups. The group of 808-NM2+4 Gy means three 4 Gy doses of ionizing radiation for a total dose of 12 Gy.
monitored by thin-layer chromatography (TLC). Flash chromatog-
raphy was carried out using silica gel (300−400 mesh). H NMR
filtered. The filtration was concentrated, and the residue was purified by
flash chromatography (EtOAc/hexane: 1/2) to give a white solid of 3a
(1.77 g, yield 38%), 1H NMR δ 7.203 (d, J = 8.0 Hz, 1H), 7.185 (d, J =
8.0 Hz, 1H), 4.613 (t, J = 8.0 Hz, 2H), 3.360 (t, J = 8.0 Hz, 2H), 2.406
(m, J = 8.0 Hz, 2H). 3b (2.59 g, yield 47%), 1 H NMR δ 7.133 (d, J = 4.0
Hz, 1H), 7.079 (d, J = 4.0 Hz, 1H), 4.404 (t, J = 8.0 Hz, 2H), 3.389 (t, J
= 8.0 Hz, 2H), 1.889−1.354 (m, 8H). 3c (3.06 g, yield 62%), 1H NMR
δ 7.724 (d, J = 8.0 Hz, 1H), 4.142 (t, J = 8.0 Hz, 2H), 3.381 (t, J = 8.0
Hz, 2H), and 2.462 (s, 3H), 2.327−2.305 (m, 2H).
Synthesis of 5a−5c. 2,2,3-Trimethyl-3H-indolenine (30 mmol)
and different bromo-substituted nitroimidazole compounds (30 mmol)
were mixed in 1,2-dichlorobenzene (15 mL) and stirred at 110 °C
under argon protection for 10−12 h. The reaction mixture was cooled
to room temperature and added to acetone. The brown precipitate or
oil was collected and washed with acetone. Indolenine quaternary
ammonium salts 5a−5c were obtained and directly used in this next
reaction without further purification.
Synthesis of 808-Nitroimidazole Conjugates. Quaternary
ammonium salts 5a, 5b or 5c (7.1 mmol) were mixed with 6 (3.3
mmol) in toluene/n-butanol [7:3 (v/v), 100 mL]. The mixture was
heated under reflux for 4−6 h. After completion of the reaction, toluene
and n-butanol were evaporated under reduced pressure to yield a yellow
green solid. The crude material was dissolved in chloroform (50 mL)
and precipitated with excess ether (150 mL). Further purification was
performed with column chromatography using dichloromethane/
methanol (15:1) as the eluent. Eventually, a green powder of each 808-
1
spectra were recorded with a Varian 400 MHz with trimethylchlor-
osilane (TMS) as an internal standard. High-resolution mass
spectrometry (HRMS) was performed on a Bruker BioTOF IIIQ
system. NIR spectroscopy was performed using an NIR spectropho-
tometer (Shimadzu, UV-3600). Photoluminescence was determined
with 740 nm excitation and scanning the wavelength from 750 to 900
nm using an NIR fluorescence spectrometer (Lumina Fluorescence
Spectrometer, Thermo Fisher USA). The purity of the final compounds
was determined by high-performance liquid chromatography (HPLC).
The purity of all final compounds was 95% or higher. The instrument
was a Shimadzu LC-6A with two pumps (LC-6AD) and a UV−VIS
detector (SPD-20AV). The column was Inertsil ODS-SP C18, 5 μm,
4.6 × 150 mm2. Eluent A was CH3CN, 0.1% TFA; eluent B was H2O,
0.1% trifluoroacetic acid (TFA); flow rate was 1 mL/min, gradual
eluent A from 0 to 95% for 11 min. The column temperature was room
temperature.
Synthesis of 3a−3c. To a mixture of 2-nitroimidazole or 5-nitro-2-
methyl-imidazole (20 mmol) and potassium carbonate (20 mmol, 2.76
g) in 15 mL of anhydrous dimethylformamide (DMF) was added 1,3-
dibromopropane (2 mmol, 400 mg for 3a or 3c) or 1,6-dibromohexane
(2 mmol, 484 mg, for 3b) and then stirred at 60 °C until TLC
monitoring was completed. The mixture was then diluted with 100 mL
of ethyl acetate and washed with water (20 mL × 3) as well as brine (20
mL × 3). The organic layer was dried by anhydrous sodium sulfate and
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nitroimidazole conjugate was obtained. 808-NM1 (0.50 g, yield 18%,
HPLC purity analysis 99.3%). 1H NMR (400 Hz, CDCl3): 8.25 (d, J =
8.0 Hz, 2H); 7.74 (S, 2H); 7.62 (d, J = 8.0 Hz, 2H); 7.45−7.42 (m,
4H); 7.27 (t, J = 4.0 Hz, 2H); 7.19 (s, 2H); 6.24 (d, J = 12.0 Hz, 2H);
4.53 (t, J = 4.0 Hz, 4H); 4.31 (J = 4.0 Hz, 4H); 2.65 (t, J = 4.0 Hz, 4H);
2.28−2.25 (m, 4H), 1.84−1.83 (m, 2H); and 1.66 (s, 12H). 13 C NMR
(100 Hz, dimethyl sulfoxide (DMSO)-d6): 172.74, 148.71, 145.07,
143.63, 142.38, 141.50, 129.05, 128.48, 127.89, 126.92, 125.71, 123.03,
111.76, 102.08, 49.51, 47.22, 41.55, 28.34, 27.91, 26.27, 21.50, and
20.78. HRMS [M − Br]+: calculated 761.3325, found 761.3354. 808-
NM2 (0.79 g, yield 26%, HPLC purity analysis 99.4%). 1H NMR (400
Hz, CDCl3): 8.22 (d, J = 8.0 Hz, 2H); 7.67 (s, 2H); 7.61 (d, J = 2 Hz,
2H); 7.43−7.40 (m, 4H); 7.27−7.25 (m, 2H); 7.15 (s, 2H); 7.29 (d, J =
6.0 Hz, 2H); 4.34 (t, J = 8.0 Hz, 4H); 4.18 (t, J = 4.0 Hz, 4H); 2.66 (t, J
= 4.0 Hz, 4H); 1.83−1.82 (m, 2H); 1.76−1.69 (m, 8H); 1.64 (s, 12H);
1.42−1.39 (m, 4H); and 1.33−1.30 (m, 4H). 13C NMR (100 Hz,
DMSO-d6): 172.60, 148.41, 144.96, 143.41, 142.49, 141.50, 129.06,
128.29, 128.27, 126.57, 125.61, 122.98, 111.97, 102.01, 49.75, 49.44,
44.17, 30.06, 27.91, 27.27, 26.25, 26.08, 25.99, 21.51, and 20.80. HRMS
[M − Br]+: calculated 845.4264, found 845.4262. 808-NM3 (0.50 g,
yield 18%, HPLC purity analysis 99.5%). 1H NMR (400 Hz, CDCl3):
8.45 (s, 2H); 8.22 (d, J = 8.0 Hz, 2H); 7.62 (s, 1H); 7.61 (s, 1H); 7.47
(d, J = 8.0 Hz, 2H); 7.42 (t, J = 4.0 Hz, 2H); 7.28 (t, J = 4.0 Hz, 2H);
6.14 (d, J = 8.0 Hz, 2H); 4.24 (d, J = 4 Hz, 4H); 4.17 (d, J = 4 Hz, 4H);
2.59 (d, J = 4.0 Hz, 4H); 2.37 (s, 12H); 2.22−2.20 (m, 4H); 1.79−1.77
(m, 2H); and 1.67 (s, 12H). 13C NMR (100 Hz, DMSO-d6): 172.58,
148.63, 145.98, 145.57, 143.54, 142.30, 141.47, 129.05, 126.85, 125.72,
123.02, 122.25, 111.85, 101.97, 49.49, 44.25, 41.54, 27.90, 27.79, 26.25,
20.83, and 13.19. HRMS [M − Br]+: calculated 789.3638, found
789.3661.
Cell Uptake and Subcellular Location. The cellular uptake of
808-NM2 was imaged and determined among MCF-10A, MCF-7, and
4T1 cells by laser confocal microscopy and flow cytometry analysis. All
cells (5 × 105 cells/well) were seeded into six-well plates and cultured
for 24 h. Cells were incubated in 1.25 μM 808-NM2 for 30 min and then
stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime,
Shanghai) for 10 min. After washing with phosphate-buffered saline
(PBS) three times, cells were imaged using a Leica DMI6000 inverted
microscope with a Leica TCS SP5 confocal scanning system
(excitation: 808 nm channel). For flow cytometry analysis, the cells
were trypsinized and collected with PBS. Cellular fluorescence was
measured on a flow cytometer using an Accuir C6 flow cytometer (BD,
Ann Arbor, MI). The cellular uptake of 808-NM2 was compared in
MCF-7 and 4T1 cells in a normoxia chamber (21% O2) with a CO2
incubator (Thermo Fisher Scienticfic, CO2 Incubator Steri-Cycle), a
hypoxic chamber (5% O2) with a tri-gas incubator (Thermo Fisher
Scienticfic, CO2 Incubator 3131) or an anaerobic incubator (Thermo
Fisher Scienticfic, Forma 1029) under hypoxia (1% O2), respectively.
MCF-7 and 4T1 cells were seeded into six-well plates at a density of 5 ×
105 cells/well in 2 mL complete medium and cultured at 37 °C for 24 h.
Cells were incubated with 1.25 μM 808-NM2 for 30 min at 37 °C. After
washing three times with PBS, cells were stained with DAPI for 10 min.
Then, the cells were collected and washed three times with PBS.
Cellular fluorescence was observed by laser confocal microscopy or
measured by flow cytometry analysis. For subcellular localization,
MCF-7 and 4T1 cells (5 × 105 cells/well) were seeded into six-well
plates and cultured for 24 h. All cells were incubated with 1.25 μM 808-
NM2 for 1 h, followed by MitoTracker Green (M7514) for 25 min.
Then, the cells were fixed with 4% paraformaldehyde and stained with
DAPI for 10 min in the dark. Finally, 1 mL 70% glycerinum was added
to each well and imaged by confocal microscopy.
nm. The cell viabilities of MCF-7 and 4T1 cells were compared under
hypoxic and normoxic conditions during incubation with 808-NM2.
MCF-7 and 4T1 cells (3 × 103 cells/well) were cultured with 100 μL of
medium in 96-well plates for 24 h. The cells were cultured with different
concentrations of 808-NM2 in a hypoxic chamber (5% O2, 5% CO2) or
normoxia chamber (5% CO2) for 24 h. The cell viabilities of MCF-7
and 4T1 cells were then tested by the MTT method as mentioned
above. To observe multimodel anticancer activities, the cell viabilities of
MCF-7 and 4T1 cells were determined after exposure to ionizing
radiation under hypoxic and normoxic conditions. MCF-7 and 4T1
cells (3 × 103 cells/well) were cultured with 100 μL of medium in 96-
well plates for 24 h. The cells were cultured with different
concentrations of 808-NM2 in a hypoxic chamber (5% O2, 5% CO2)
for 6 h. Then, the cells were treated with ionizing radiation (1 and 3 Gy)
using RS2000 X-ray irradiator (RadSource Technologies) and
incubated for 72 h. The cell viabilities were tested by MTT method
as mentioned above.
Animals and Tumor Xenografts. Female BALB/c nude mice (4−
6 weeks old) were purchased from the laboratory animal center of Third
Military Medical University. All animal studies were approved by the
Ethics Committee of the Third Military Medical University
(Chongqing, China). For the tumor growth assay, nude mice were
injected with 4T1 cells in 200 μL of serum-free RPMI-1640 medium at
2 × 105 cells/mouse in the left flank. Mice were randomized into three
groups and treated with tail vein injection of normal saline or 10 mg/kg
808-NM2. The next day, for the 808-NM2 radiation group, only tumors
were exposed to 4 Gy ionizing radiation with a lead cover. Small animal
biological irradiator (Pxi X-RAD 320) was used for ionizing radiation
treatment. Using a lead cover, only tumors were exposed to X-ray
irradiation. Every other day, the mice were treated with 808-NM2 and
irradiated three times. Tumor size was measured by calipers once every
2 days. On the 11th day after the first injection of 808-NM2, all of the
tumors in the different groups were dissected from the mice and
weighed. Lung organs were collected after mice were sacrificed at the
end of observation. H&E staining or immunochemical staining of
tumors or organs for pathological histology analysis was performed.
Patients-Derived Xenografts. Human samples were obtained
from all patients with written informed consent. Both written informed
consent and the study protocol were approved by the ethics committee
of the First Affiliated Hospital of Army Medical University (namely,
Southwest Hospital, Third Military Medical University, Approved No.
KY2020002). Breast cancer fragments (0.5 cm) were obtained from the
Department of Oncology, Southwest Hospital, Third Military Medical
University and implanted in the left flanks of nude mice. Twenty days
after tumor implantation, mice were divided into three groups: the
control group, 808-NM2 (10 mg/kg) group, and 808-NM2 irradiation
group (4 Gy). For the control and 808-NM2 groups, mice were injected
with saline or 10 mg/kg 808-NM2, respectively. The next day, mice in
the 808-NM2 radiation group were exposed to 4 Gy ionizing radiation.
Every other day, the mice were treated with 808-NM2 and irradiated
three times. Tumor size was measured by calipers once every 2 days.
The mouse body weight was also recorded during treatment. On the
11th day after the first injection of 808-NM2, all tumors in the different
groups were dissected from the mice and further stained for different
kinds of immunohistochemistry analysis.
In Vivo NIR Imaging. In vivo NIR imaging was performed using a
small animal imaging system (Perkin Elmer, IVIS Spectrum). For the
assessment of the tumor-targeting ability of 808-nitroimidazole
conjugates, BALB/c mice bearing 4T1 xenografts were established
and injected at a dose of 0.5 mg/kg by tail vein. In vivo NIR imaging was
performed at different times after injection. After 24 h, mice were
sacrificed, and the main organs, including tumors, were collected. Ex
vivo NIR imaging was performed to observe their distribution. To assess
the tumor-targeting ability in the PDX model, athymic nude mice were
injected with 808-NM2 conjugates at a dose of 0.5 mg/kg via the tail
vein. For comparison, the same protocols were performed, but nothing
was implanted in mice as a sham-operated group.
In Vitro Anticancer Effect. The cytotoxicity of MCF-7 and 4T1
cells (3000 cells/well) was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT, M5655, Sigma-Aldrich)
assay. MCF-7 and 4T1 cells were cultured with 100 μL of medium in
96-well plates and cultured for 24 h. Then, the cells were incubated with
different concentrations of 808-NM2 for 72 h at 37 °C. The solution
was discarded. Then, 20 μL of MTT solution (5 mg/mL) was added to
each well and incubated for 4 h at 37 °C. The crystals were dissolved in
200 μL of DMSO, and the optical density (O.D.) was recorded at 490
Statistics Analysis. Data were reported as the mean and standard
error of the mean (SEM) from at least three independent experiments;
the p value was evaluated and calculated using a two-tailed paired t-test.
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Article
When there were multiple factors involved, a two-way analysis of
variance (ANOVA) was used; multiple groups were compared using a
one-way or two-way ANOVA. Values of p < 0.05 (* or #) were
considered statistically significant.
Shuang Long − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China
Jing Liu − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China
Yu Lan − Department of Radiology, Athinoula A. Martinos
Center for Biomedical Imaging, Massachusetts General
Hospital, Harvard Medical School, Charlestown,
Massachusetts 02129, United States
Dong Yang − Department of Radiology, Athinoula A. Martinos
Center for Biomedical Imaging, Massachusetts General
Hospital, Harvard Medical School, Charlestown,
Massachusetts 02129, United States
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02250.
Additional experimental methods; synthetic route of 808-
NM1, 808-NM2, and 808-NM3 (Figures S1 and S2);
structural characterization of 808-NM2 (Figures S3 and
S4); purity of final compounds (Figure S5); NIR optical
determination (Figure S6); in vitro and in vivo tests on
tumor growth inhibition (Figures S7−S23); physico-
chemical properties of 808-nitroimidazole conjugates
(Table S1); and patient characteristics for the PDX
model (Table S2) (PDF)
Molecular formula strings (CSV)
Hao Wang − Department of Radiology, Athinoula A. Martinos
Center for Biomedical Imaging, Massachusetts General
Hospital, Harvard Medical School, Charlestown,
Massachusetts 02129, United States
AUTHOR INFORMATION
Corresponding Authors
■
Changning Wang − Department of Radiology, Athinoula A.
Martinos Center for Biomedical Imaging, Massachusetts
General Hospital, Harvard Medical School, Charlestown,
Massachusetts 02129, United States; orcid.org/0000-
0002-2076-4193; Email: cwang15@mgh.harvard.edu
Shenglin Luo − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China; orcid.org/0000-0002-0804-9891; Email: luosl@
tmmu.edu.cn
Shuhui Li − Department of Clinical Biochemistry, Laboratory
Sciences, Third Military Medical University (Army Medical
University), Chongqing 400038, People’s Republic of China
An Chen − Department of Clinical Biochemistry, Laboratory
Sciences, Third Military Medical University (Army Medical
University), Chongqing 400038, People’s Republic of China
Yuhui Hao − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China
Yongping Su − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China
Authors
Sha Chen − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine and Department of Clinical Biochemistry,
Laboratory Sciences, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02250
Songtao Yu − Department of Oncology, Southwest Hospital,
Third Military Medical University (Army Medical University),
Chongqing 400038, People’s Republic of China; orcid.org/
0000-0002-2804-9581
Zaizhi Du − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China
Xie Huang − State Key Laboratory of Trauma, Burns and
Combined Injury, Institute of Combined Injury, Chongqing
Engineering Research Center for Nanomedicine, College of
Preventive Medicine, Third Military Medical University (Army
Medical University), Chongqing 400038, People’s Republic of
China
Meng He − Department of Clinical Biochemistry, Laboratory
Sciences, Third Military Medical University (Army Medical
University), Chongqing 400038, People’s Republic of China
Author Contributions
⊥S.C. and S.Y. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors of this work acknowledge the financial support by
National Natural Science Foundation of China (Nos. 81773352
and 81402784), Chongqing Youth Talent Project
(CQYC2020058690), China Scholarship Council (No.
201703170071), Outstanding Youth Development Program in
Third Military Medical University (No. 2017MPRC-17), and
Intramural Research Project Grant (AEP17J001).
ABBREVIATIONS
BSP, sulfobromophthalein; 2-DG, 2-deoxy-D-glucose; mtDNA,
mitochondrial DNA; NIR, near infrared; OATP, organic anion
■
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(17) Lv, Q. Z.; Yang, X. G.; Wang, M. L.; Yang, J. C.; Qin, Z. L.; Kan,
Q. M.; Zhang, H. T.; Wang, Y. J.; Wang, D.; He, Z. G. Mitochondria-
targeted prostate cancer therapy using a near-infrared fluorescence dye-
monoamine oxidase A inhibitor conjugate. J. Controlled Release 2018,
279, 234−242.
transporting polypeptide; PET, positron emission tomography;
PDX, patient-derived xenograft; ROS, reactive oxygen species;
SPECT, single-photon emission computed tomography;
VEGFR2, vascular endothelial growth factor receptor 2
(18) Luo, S.; Tan, X.; Fang, S. T.; Wang, Y.; Liu, T.; Wang, X.; Yuan,
Y.; Sun, H. Q.; Qi, Q. R.; Shi, C. M. Mitochondria-targeted small-
molecule fluorophores for dual modal cancer phototherapy. Adv. Funct.
Mater. 2016, 26, 2826−2835.
(19) Tan, X.; Luo, S. L.; Long, L.; Wang, Y.; Wang, D. C.; Fang, S. T.;
Ouyang, Q.; Su, Y. P.; Cheng, T. M.; Shi, C. M. Structure-guided design
and synthesis of a mitochondria-targeting near-infrared fluorophore
with multimodal therapeutic activities. Adv. Mater. 2017, 29,
No. 1704196.
(20) Ntziachristos, V. Going deeper than microscopy: the optical
imaging frontier in biology. Nat. Methods 2010, 7, 603−614.
(21) Guo, Q.; Luo, S.; Qi, Q.; Shi, C. Preliminary structure-activity
relationship study of a couple of heptamethine indocyanine dyes for
tumor-targeted imaging. J. Innovative Opt. Health Sci. 2013, 6,
No. 1350003.
REFERENCES
■
(1) Wilson, W. R.; Hay, M. P. Targeting hypoxia in cancer therapy.
Nat. Rev. Cancer 2011, 11, 393−410.
(2) Barker, H. E.; Paget, J. T.; Khan, A. A.; Harrington, K. J. The
tumour microenvironment after radiotherapy: mechanisms of resist-
ance and recurrence. Nat. Rev. Cancer 2015, 15, 409−425.
(3) Pettersen, E.; Ebbesen, P.; Gieling, R.; Williams, K.; Dubois, L.;
Lambin, P.; Ward, C.; Meehan, J.; Kunkler, I.; Langdon, S.; Ree, A.;
Flatmark, K.; Lyng, H.; Calzada, M.; Peso, L.; Landazuri, M.; Görlach,
A.; Flamm, H.; Kieninger, J.; Urban, G.; Weltin, A.; Singleton, D. C.;
Haider, S.; Buffa, F. M.; Harris, A. L.; Scozzafava, A.; Supuran, C. T.;
Moser, I.; Jobst, G.; Busk, M.; Toustrup, K.; Overgaard, J.; Alsner, J.;
Pouyssegur, J.; Chiche, J.; Mazure, N.; Marchiq, I.; Parks, S.; Ahmed, A.;
Ashcroft, M.; Pastorekova, S.; Cao, Y.; Rouschop, K. M.; Wouters, B.
G.; Koritzinsky, M.; Mujcic, H.; Cojocari, D. Targeting tumour hypoxia
to prevent cancer metastasis. From biology, biosensing and technology
to drug development: the METOXIA consortium. J. Enzyme Inhib. Med.
Chem. 2015, 30, 689−721.
(22) Zhang, E.; Luo, S.; Tan, X.; Shi, C. Mechanistic study of IR-780
dye as a potential tumor targeting and drug delivery agent. Biomaterials
2014, 35, 771−778.
(23) Kroemer, G.; Reed, J. C. Mitochondrial control of cell death. Nat.
Med. 2000, 6, 513−519.
(4) Forster, J. C.; Marcu, L. G.; Bezak, E. Approaches to combat
hypoxia in cancer therapy and the potential for in silico models in their
evaluation. Phys. Med. 2019, 64, 145−156.
(5) Jahanban-Esfahlan, R.; de la Guardia, M.; Ahmadi, D.; Yousefi, B.
Modulating tumor hypoxia by nanomedicine for effective cancer
therapy. J. Cell. Physiol. 2018, 233, 2019−2031.
(6) Ikeda, Y.; Hisano, H.; Nishikawa, Y.; Nagasaki, Y. Targeting and
treatment of tumor hypoxia by newly designed prodrug possessing high
permeability in solid tumors. Mol. Pharm. 2016, 13, 2283−2289.
(7) Liang, D.; Miller, G. H.; Tranmer, G. K. Hypoxia activated
prodrugs:Factors influencing design and development. Curr. Med.
Chem. 2015, 22, 4313−4325.
(8) Bonnet, M.; Hong, C. R.; Wong, W. W.; Liew, L. P.; Shome, A.;
Wang, J.; Gu, Y.; Stevenson, R. J.; Qi, W.; Anderson, R. F.; Pruijn, F. B.;
Wilson, W. R.; Jamieson, S. M. F.; Hicks, K. O.; Hay, M. P. Next-
generation hypoxic cell radiosensitizers: nitroimidazole alkylsulfona-
mides. J. Med. Chem. 2018, 61, 1241−1254.
(9) Meißner, R.; Kocisek, J.; Feketeova, L.; Fedor, J.; Farnik, M.;
Limao-Vieira, P.; Illenberger, E.; Denifl, S. Low-energy electrons
transform the nimorazole molecule into a radiosensitiser. Nat. Commun.
2019, 10, No. 2388.
(10) Overgaard, J. Hypoxic Radiosensitization: adored and ignored. J.
Clin. Oncol. 2007, 25, 4066−4074.
(11) Tan, X.; Luo, S.; Wang, D.; Su, Y.; Cheng, T.; Shi, C. A NIR
heptamethine dye with intrinsic cancer targeting, imaging and
photosensitizing properties. Biomaterials 2012, 33, 2230−2239.
(12) Shi, C.; Zhang, C.; Su, Y.; Cheng, T. Cyanine dyes in optical
imaging of tumours. Lancet Oncol. 2010, 11, 815−816.
(13) Duan, L. L.; Wang, L.; Zhang, C.; Yu, L.; Guo, F.; Sun, Z.; Xu, Y.;
Yan, F. Role of near-infrared heptamethine cyanine dye IR-783 in
diagnosis of cervical cancer and its mechanism. Int. J. Clin. Exp. Pathol.
2019, 12, 2353−2362.
(24) Wallace, D. C. Mitochondria and cancer. Nat. Rev. Cancer 2012,
12, 685−698.
(25) Shi, C. H.; Wu, J. B.; Chu, G. C. Y.; Li, Q. L.; Wang, R. X.; Zhang,
C. Q.; Zhang, Y.; Kim, H. L.; Wang, J.; Zhau, H. Y. E.; Pan, D. F.;
Chung, L. W. K. Heptamethine carbocyanine dye-mediated near-
infrared imaging of canine and human cancers through the HIF-1
alpha/OATPs signaling axis. Oncotarget 2014, 5, 10114−10126.
(26) Wu, J. B.; Shao, C.; Li, X.; Shi, C.; Li, Q.; Hu, P.; Chen, Y.-T.;
Dou, X.; Sahu, D.; Li, W.; Harada, H.; Zhang, Y.; Wang, R.; Zhau, H. E.;
Chung, L. W. K. Near-infrared fluorescence imaging of cancer mediated
by tumor hypoxia and HIF1α/OATPs signaling axis. Biomaterials 2014,
35, 8175−8185.
(27) Lee, W.; Belkhiri, A.; Lockhart, A. C.; Merchant, N.; Glaeser, H.;
Harris, E. I.; Washington, M. K.; Brunt, E. M.; Zaika, A.; Kim, R. B.; El-
Rifai, W. Overexpression of OATP1B3 confers apoptotic resistance in
colon cancer. Cancer Res. 2008, 68, 10315−10323.
(28) Franken, N. A. P.; Rodermond, H. M.; Stap, J.; Haveman, J.; van
Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 2006, 1, 2315−
2319.
(29) Yu, C. Y.; Xu, H.; Ji, S.; Kwok, R. T.; Lam, J. W.; Li, X.; Tang, B.
Z.; et al. Mitochondrion-anchoring photosensitizer with aggregation-
induced emission characteristics synergistically boosts the radio-
sensitivity of cancer cells to ionizing radiation. Adv. Mater. 2017, 29,
No. 1606167.
(30) Baracca, A.; Sgarbi, G.; Solaini, G.; Lenaz, G. Rhodamine 123 as a
probe of mitochondrial membrane potential: evaluation of proton flux
through F0 during ATP synthesis. Biochim. Biophys. Acta 2003, 1606,
137−146.
(31) Ishikawa, K.; Takenaga, K.; Akimoto, M.; Koshikawa, N.;
Yamaguchi, A.; Imanishi, H.; Nakada, K.; Honma, Y.; Hayashi, J. ROS-
Generating mitochondrial DNA mutations can regulate tumor cell
metastasis. Science 2008, 320, 661−664.
(32) Tuma, R. S. Making ROS for apoptosis. J. Cell Biol. 2005, 170,
513.
(14) Xiao, L.; Zhang, Y.; Yue, W.; Xie, X. Z.; Wang, J. P.; Chordia, M.
D.; Chung, L. W. K.; Pan, D. F. Heptamethine cyanine based Cu-64-
PET probe PC-1001 for cancer imaging: Synthesis and in vivo
evaluation. Nucl. Med. Biol. 2013, 40, 351−360.
(33) Hoeben, A.; Landuyt, B.; Highley, M. S.; Wildiers, H.; Van
Oosterom, A. T.; De Bruijn, E. A. Vascular endothelial growth factor
and angiogenesis. Pharmacol. Rev. 2004, 56, 549−580.
(34) Guru, S. K.; Pathania, A. S.; Kumar, S.; Ramesh, D.;
Vishwakarma, R. A.; Bhushan, S.; et al. Secalonic Acid-D Represses
HIF1 alpha/VEGF-Mediated Angiogenesis by Regulating the Akt/
mTOR/p70S6K Signaling Cascade. Cancer Res. 2015, 75, 2886−2896.
(35) Sullivan, H. C.; Edgar, M. A.; Cohen, C.; Kovach, C. K.; HooKim,
K.; Reid, M. D. The utility of ERG, CD31 and CD34 in the cytological
(15) Zhang, Y.; Xiao, L.; Popovic, K.; Xie, X. Z.; Chordia, M. D.;
Chung, L. W. K.; Williams, M. B.; Yue, W.; Pan, D. F. Novel cancer-
targeting SPECT/NIRF dual-modality imaging probe 99m Tc-PC-
1007: Synthesis and biological evaluation. Bioorg. Med. Chem. Lett.
2013, 23, 6350−6354.
(16) Wu, J. B.; Lin, T. P.; Gallagher, J. D.; Kushal, S.; Chung, L. W. K.;
Zhau, H. Y. E.; Oenyuk, B. Z.; Shih, J. C. Monoamine oxidase A
inhibitor-near-infrared dye conjugate reduces prostate tumor growth. J.
Am. Chem. Soc. 2015, 137, 2366−2374.
3390
https://dx.doi.org/10.1021/acs.jmedchem.0c02250
J. Med. Chem. 2021, 64, 3381−3391

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
diagnosis of angiosarcoma: an analysis of 25 cases. J. Clin. Pathol. 2015,
68, 44−50.
3391
https://dx.doi.org/10.1021/acs.jmedchem.0c02250
J. Med. Chem. 2021, 64, 3381−3391