Hypoxia-Activated and Indomethacin-Mediated Theranostic Prodrug Releasing Drug On-Demand for Tumor Imaging and Therapy

Article abstract of DOI:10.1021/acs.bioconjchem.9b00564

A smart theranostic prodrug IMC-FDU-TZBC-NO₂, releasing active drug on-demand based on hypoxia-activated and indomethacin-mediated, for solid tumor imaging and efficient therapy was designed. This prodrug was constructed by conjugating chemotherapy drug 5-fluoro-2-deoxyuridine (FDU), targeting moiety indomethacin (IMC), and the hypoxic trigger 4-nitrobenzyl group to a fluorescent dye precursor, which was mediated by IMC and activated by NTR under hypoxic conditions. The fluorescent dye IMC-TZBCM was generated and FDU was released at the same time in tumor cells. The rates and amounts of FDU release and IMC-TZBCM generation were regulated by hypoxia status, and increased with increasing degree of hypoxia. Nevertheless, it is "locked" in normal cells. It combined the advantages of tumor targeting, diagnosis, and chemotherapy functions, showed excellent targeting ability to cancer cells, excellent stability in physiological conditions, high cellular uptake efficiency, and on-demand drug release behavior. The in vitro and in vivo assays demonstrated that IMC-FDU-TZBC-NO₂ exhibits enhanced anticancer potency and low side effects. The novel targeted theranostic prodrug activated by hypoxia shows a great potential in cancer therapy.

Full text of DOI:10.1021/acs.bioconjchem.9b00564

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Article
Hypoxia-activated and indomethacin-mediated theranostic
prodrug releasing drug on-demand for tumor imaging and therapy
Xiaoran Peng, Jiaxin Gao, Yumeng Yuan, Haitong Liu,
Weiyan Lei, Shenghui Li, Jinchao Zhang, and Shuxiang Wang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00564 • Publication Date (Web): 08 Oct 2019
Downloaded from pubs.acs.org on October 13, 2019
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Graphical abstract
Hypoxia-activated and indomethacin-mediated theranostic prodrug releasing drug
on-demand for tumor imaging and therapy
Xiaoran Peng, Jiaxin Gao, Yumeng Yuan, Haitong Liu, Weiyan Lei, Shenghui Li, Jinchao Zhang, and Shuxiang Wang
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Hypoxia-Activated and Indomethacin-Mediated Theranostic Prodrug
Releasing Drug on-Demand for Tumor Imaging and Therapy
a
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a,b
Xiaoran Peng, Jiaxin Gao, Yumeng Yuan, Haitong Liu, Weiyan Lei, Shenghui Li,
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Jinchao Zhang, * and Shuxiang Wang*
aKey Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei
University, Baoding 071002, China
bKey Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental
Science, Hebei University, Baoding 071002, China
Email: jczhang6970@163.com; wsx@hbu.edu.cn
Supporting Information
ABSTRACT: A smart theranostic prodrug IMC-FDU-TZBC-NO , releasing active drug on-demand
2
based on hypoxia-activated and indomethacin-mediated, for solid tumor imaging and efficient
therapy was designed. This prodrug was constructed by conjugating of chemotherapy drug
5
-fluoro-2-deoxyuridine (FDU), targeting moiety indomethacin (IMC), and hypoxic trigger
4-nitrobenzyl group to a fluorescent dye precursor, which was mediated by IMC and activated by
NTR under hypoxia conditions. The fluorescent dye IMC-TZBCM was generated and FDU was
released at the same time in tumor cells. The rates and amounts of FDU release and IMC-TZBCM
generation were regulated by hypoxia status, and increased with increasing degree of hypoxia.
Nevertheless, it is "locked" in normal cells. It combined the advantages of tumor targeting,
diagnosis, and chemotherapy functions together, showed excellent targeting ability to cancer
cells, excellent stability in physiological conditions, high cellular uptake efficiency, and
on-demand drug release behavior. The in vitro and in vivo assays demonstrated that
IMC-FDU-TZBC-NO exhibiting enhanced anticancer potency and low side effect. The novel
2
targeted theranostic prodrug activated by hypoxia shows a great potential in cancer therapy.
■
INTRODUCTION
1
In recent years, the incidence of cancer and death rates have continued to rise, and
2
chemotherapy is a main stream approach for cancer therapy. Despite several anticancer drugs
such as doxorubicin (DOX), paclitaxel, camptothecin (CPT), fluorouracil, and gemcitabine were
approved by the FDA, it is a formidable challenge to achieve the desired therapeutic effect with
minimum side effects, since these chemotherapy strategies are based on high doses and poor
3
selectivity for tumor cells, which results in side effects and drug resistance. Therefore,
considerable efforts have been devoted to explore efficient strategies of prodrugs, aiming at
improving anticancer drugs specifically into tumors and on-demand releasing with controlled
4
temporal profile to enhance therapeutic efficacy and reduce side effects. In particular,
theranostic agents with both therapeutic and diagnostic functions are attracting great interest
due to their reduced side effects, and enhanced clinical efficacy.5-8 Theses theranostic agents
were triggered by specific internal or external stimulus (such as high acidity,9-14 over-expressed
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enzymes,15-22 reactive oxygen species,23-28 heat,29, 30 and light,31-33 etc.), and have been developed
for releasing drugs on demand inside the tumor and real-time tracking of drug release and
localization. Among these stimulation ways, over-expressed enzymes-responsive theranostic
agents have drawn great attention because of this strategy seeks to treat cancer through the
selective identification and therapeutics in cancer tissue.
Hypoxia as a common feature of all solid tumors is associated with the tumor growth,34, 35
angiogenesis,36 invasion,37 angiogenesis, recurrence, and metastatsis.38 Therefore, hypoxia is an
indicator of poor prognosis and therapeutic outcome, resistance to chemotherapy, and
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recurrence. However, hypoxia also provides a hopefully useable target for cancer diagnosis and
treatment. In hypoxic solid tumors, nitroreductase (NTR) is over-expressed, which can be used as
trigger for design selective anticancer prodrug. Therefore, hypoxia activation provides a pathway
for hypoxia-activated prodrug to release drug on-demand. A number of prodrugs activated by
NTR under hypoxia conditions have been developed to improve tumor-selective
chemotherapeutic effects.40-43 TH-302 as a hypoxia-activated prodrug showed promise
therapeutic effect in preclinical and phase II trials.44 However, it was reported that TH-302 did
not achieve primary overall survival endpoints in combination with chemotherapy in two large
phase III studies.45 Thus, targeting and controllable release of active drug at tumor sites is a
challenge for tumor therapy.
Cyclooxygenase (COX) is a key enzyme that plays a decisive role in catalyzing transformation of
arachidonic acid to prostaglandins, which in turn contributes to inflammation and tumorigenesis
46
in humans. Indeed, COX-2 over-expression and antiproliferative effects of COX-2 inhibitors have
been reported in various cancers (such as pancreatic, colon cancer, liver, and gastric cancers).47-50
And, COX-2 critically influences all stages of tumor progression from initiation to progression.
Therefore COX-2 can be used as an ideal target for tumor diagnosis and treatment.51-53 As an
inhibitor of COX-2, indomethacin (IMC) exhibited a great potential in the identification and
5
4
quantification of COX-2 in biological systems and has been used to distinguish between normal,
inflammatory, and cancer cells.52
In this work, we developed a novel theranostic prodrug, IMC-FDU-TZBC-NO , based on
2
hypoxia-activation and IMC-guiding for on-demand drug release and enhanced anticancer
potency with low side effect. In this design (Scheme 1) ， 5-fluoro-2-deoxyuridine (FDU) was
chosen as the active drug due to displaying high antitumor activity, hydrophilicity, 55, 56 and being
widely used in the treatments of cancer. IMC was used as a targeting moiety to improve the drug
targeting effect,57 cellular uptake efficiency, and drug accumulation; 4-nitrobenzyl group was
used as a trigger to hypoxia to release active drug on demand based on hypoxic status;
1
,1'-biphenyl-4'-((1H-1,2,3-triazol-4-yl)methyl)ethylamino-2'-(4-nitrobenzyloxy)-2-carboxylate
(BPBC-NO ) was used as a fluorescent dye precursor for formation a fluorescent dye IMC-TZBCM
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under hypoxia conditions; and BPBC-NO was connected with IMC, 4-nitrobenzyl group, and FDU
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by covalent bonds. The obtained IMC-FDU-TZBC-NO not only can target tumor cells, but also are
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able to release IMC-TZBCM and FDU simultaneously under hypoxic conditions for investigating
the hypoxic status, in vitro and in vivo inhibition of tumor cell growth with high efficiency and
minimal side effects. This strategy will provide a valuable tool for cancer therapy on individual
patient’s demand based on the individual hypoxic status.
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Scheme 1. Structure and Schematic Illustration of IMC-FDU-TZBC-NO2
Scheme 1
■
RESULTS AND DSISCUSSION
Synthesis and Characterization. To examine the effectiveness of this strategy, IMC-TZBCM,
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FDU-TZBC-NO , and IMC-mediated theranostic prodrug IMC-FDU-TZBC-NO were synthesized
2 2
(Scheme 2). Firstly, the ethylation reaction of 4-bromo-3-methoxyaniline with bromoethane in
dry DMF afforded compound 1. Subsequently, the demethylation of compound 1 by BBr3
afforded compound 2. Then, compound 2 was protected at the arylamine N by means of (Boc) O
2
to afford compound 3 at room temperature. Followed by nucleophilic substitution of compound
3
with 4-nitrobenzyl-bromide, t-butyl (4-bromo-3-((4-nitrobenzyl)oxy)phenyl) ethylcarbamate (4)
was generated. Subsequently, ethyl 4'-((t-butoxycarbonyl)ethylamino)-2'-
((4-nitrobenzyl)oxy)-1,1'-biphenyl-2-carboxylate (5) was synthesized via Suzuki cross-couping
reaction between compound 4 with 2-(ethoxycarbonyl)phenyl boronic acid under N2
atmosphere. Next, N-boc deprotection of compound 5 was achieved by a solution of HCl (4 M) in
anhydrous THF to afford ethyl 4'-(ethylamino)-2'-((4-nitrobenzyl)oxy)-1,1'-biphenyl-2-carboxylate
(6). Followed by nucleophilic substitution of compound 6 with 3-bromoprop-1-yne, ethyl
4
'-(ethyl(prop-2-yn-1-yl)amino)-2'-((4-nitrobenzyl)oxy)-1,1'-biphenyl-2-carboxylate (7) was
generated. Afterwards, take advantage of hydrolysis of compound 7 in sulfuric acid aqueous
solution, 4'-(ethyl(prop-2-yn-1-yl)amino)-2'-((4-nitrobenzyl)oxy)-1,1'-biphenyl-2-carboxylic acid
(8) was obtained. Then, FDU was conjugated with compound 8 to provide FDU-TZBC-NO (as the
2
control drug) using DIC as condensation agent and catalytic amount of DMAP. Meanwhile, IMC
was modified by azidopropan-1-amine via an amidation reaction in presence of HATU to afford
IMC-N . Then, IMC-TZBC-NO was generated by the click reaction of IMC-N with compound 8 at
3
2
3
2
5 °C in the presence of Vc and CuSO . Finally, IMC-FDU-TZBC-NO was constructed via an
4 2
amidation reaction of FDU with IMC-TZBC-NO in the presence of DIC and DMAP. And, in the
2
presence of Na S O , IMC-TZBC-NO translated to IMC-TZBCM smoothly at room temperature.
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The H/ C NMR and MS of IMC-TZBC-NO , IMC-TZBCM, IMC-FDU-TZBC-NO , and intermediates
2
2
1
were well characterized (Figure S1-S32, ESI†). In the H NMR of IMC-FDU-TZBC-NO , the signal at
2
δ 11.79 (s, 1 H) was attributed to -CONHCO- of uracil. The signal at δ 8.14 (t, J = 6.0 Hz, 1 H) was
attributed to –CONHCH -. The signals at δ 8.09 (d, J = 9.0 Hz, 2 H), 7.90 (s, 1 H), 7.73 (d, J = 7.2 Hz,
2
1
H), 7.68 (d, J = 8.4 Hz, 2 H), 7.64~7.61 (m, 3 H), 7.57 (t, J = 7.2 Hz, 1 H), 7.41 (d, J = 9.0 Hz, 2 H),
7.38 (t, J = 7.2 Hz, 1 H), 7.31 (d, J = 7.8 Hz, 1 H), 7.11 (d, J = 2.4 Hz, 1 H), 6.99 (d, J = 7.2 Hz, 1 H),
6
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.93 (d, J = 9.0 Hz, 1 H), 6.69 (dd, J = 7.2, 2.4 Hz, 1 H), 6.43 (dd, J = 8.4, 1.8 Hz, 1 H), 6.38 (s, 1 H),
.05 (t, J = 6.6 Hz, 1 H) displayed characteristic of substituted 1,2,3-triazole, uracil, and benzene
rings. The peak at δ 5.76 (s, 1 H) was attributed to -OH. The peak at δ 5.12 (s, 2 H) was attributed
to -O-CH -Ph-NO . Signal at δ 4.52 (s, 2 H) was attributed to –NCH -1,2,3-triazole. Signal at δ 3.72
2
2
2
(
s, 3 H) was attributed to –OCH . The peak at δ 3.50 (s, 2 H) was attributed to -CO-CH -indole.
3 2
Signals at δ 3.43 (q, J = 6.6 Hz, 2 H) and 1.07 (t, J = 6.6 Hz, 3 H) were attributed to –NCH CH .
2
3
Signal at δ 2.21 (s, 3 H) was attributed to –CH on the indole ring. The peaks at δ 5.34 (d, J = 4.2
3
Hz, 1 H), 4.30 (t, J = 7.2 Hz, 2 H), 4.22~4.16 (m, 2 H), 3.94~3.92 (m, 1 H), 3.79 (q, J = 4.2 Hz, 1 H),
3
.17 (d, J = 5.4 Hz, 1 H), 3.05 (q, J = 6.0 Hz, 2 H), and 1.92 (q, 3 H) were attributed to protons of
aliphatic methene and methine of deoxyribose.
Scheme 2. Synthetic Routes for FDU-TZBC-NO , IMC-FDU-TZBC-NO , and IMC-TZBCM
2
2
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Scheme 2
The response of IMC-FDU-TZBC-NO to NTR. To verify that NTR was able to reduce the nitro
2
group of the hypoxia trigger and consequently cleave IMC-FDU-TZBC-NO and release of active
2
drug with fluorescent dye, the absorption spectra of IMC-FDU-TZBC-NO and IMC-TZBCM were
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delineated in Figure 1A, and a transformation experiment of IMC-FDU-TZBC-NO in the presence
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of NTR/NADH under physiological conditions was performed, and then the mixture was
monitored by fluorescence spectroscopy. IMC-TZBCM displayed a significant green fluorescence
with maxima at 480 (λ =380) nm. Nevertheless, IMC-FDU-TZBC-NO solution exhibited almost no
ex
2
fluorescence signals because of the quenching effect of 4-nitrobenzyl group and the σc-c bond
free rotation (Figure 1B). However, the mixture solution of IMC-FDU-TZBC-NO with NTR/NADH
2
showed a same bright green fluorescence with 21-fold enhancement at same maxima at
λex/em=380/480 nm. Then, the catalytic efficiency of NTR was assessed by fluorescent
spectrometry. As shown in Figure 1C and 1D, fluorescence kinetic-time curves of
IMC-FDU-TZBC-NO activated by NTR (at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 min)
2
indicated that the fluorescence intensity of the mixture system containing IMC-FDU-TZBC-NO2
with NTR enhenced quickly with the prolongation of reaction time, and reached maximum when
the mixture was reacted about 12ꢀmin. These results revealed that IMC-FDU-TZBC-NO2
responded to NTRwith quickly speed and high sensitivity under hypoxia conditions.
Figure 1
Figure 1. (A) Absorbance of IMC-FDU-TZBC-NO and IMC-TZBCM (50μM). (B) Fluorescence
2
emission spectrometry were measured before and after incubation of IMC-FDU-TZBC-NO with
2
NTR/NADH (N atmosphere, incubation time: 2 h, IMC-FDU-TZBC-NO concentration: 50 μM, NTR
2
2
concentration: 10 μg/mL, NADH concentration: 2 mM; λex/em=380/480 nm). (C) Time-dependent
fluorescence spectra, and (D) intensity of IMC-FDU-TZBC-NO2 (concentration: 100 μM)
o
incubation with NTR (concentration: 10 μg/mL) and NADH (concentration: 2 mM) at 37 C under
hypoxic conditions, λex/em=380/480 nm
Mechanism of Release of Active Drug with Fluorescent Dye from IMC-FDU-TZBC-NO2
Activated by NTR. To further confirm the mechanism of IMC-FDU-TZBC-NO activated by NTR,
2
the possibility of release of FDU with IMC-TZBCM from IMC-FDU-TZBC-NO activated by Na S O
2 2 4
2
was explored. Firstly, the relative retention times of FDU, IMC-TZBCM, and IMC-FDU-TZBC-NO2
were determined by HPLC, which were 10.930, 16.450, and 22.722 min, respectively (Figure 2A,
2
B, and 2C). Then, various composition of the mixture of IMC-FDU-TZBC-NO reaction with
2
Na S O at room temperature were analysed by comparing the retention times of various
2
2 4
components. The peak intensity at 22.715 min corresponding to the IMC-FDU-TZBC-NO was
2
decreased, and two new strong peaks at 10.913 and 16.449 min (Figure 2D) were found, which
were consistent with FDU and IMC-TZBCM matches well. These results provided an evidence for
the release of FDU and IMC-TZBCM from the prodrug IMC-FDU-TZBC-NO . Based on above
results and combined with reports,42, 59, 60 the process of releasing FDU with IMC-TZBCM from
IMC-FDU-TZBC-NO is depicted as follows: firstly, under the action of Na S O the -NO group
2
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2
2
4
2
was converted to -NH , and IMC-FDU-TZBC-NO was reduced to IMC-FDU-TZBC-NH . Immediately
2
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followed by a series of electron transfer process and the 1,6-rearrangement-elimination reaction,
small molecule 4-methylenecyclohexa- 2,5-dien-1-iminium was eliminated, and lead to
IMC-FDU-TZBC being produced. Finally, under the actions of σc-c bond rotation of diphenyl and
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intra-molecular esterification, fluorescent dye IMC-TZBCM was generated as FDU was released
simultaneously (Figure 2E).
Figure 2
Figure 2. HPLC profiles of FDU(A), IMC-FDU-TZBC-NO (B), IMC-TZBCM (C), and a mixture of
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IMC-FDU-TZBC-NO with Na S O in DMSO/H O (1/1, V/V) was stirred at 25 C for 30 min (D), the
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mechanism of IMC-FDU-TZBC-NO releasing FDU and IMC-TZBCM activated by hypoxia (E)
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The Properties of IMC-FDU-TZBC-NO2. To investigate conversion efficiency of
IMC-FDU-TZBC-NO activated by NTR, we determined the amount of released FDU from
2
IMC-FDU-TZBC-NO by HPLC method, which was incubated with NTR for different time under N2
2
atmosphere. As shown in Figure 3A, with the increasing of incubation time, the amount of FDU
increased correspondingly. In addition, after incubation for 12 min, the FDU release efficiency
reached 73%. These results revealed that IMC-FDU-TZBC-NO released FDU quickly under
2
hypoxic conditions.
To evaluate the possible application of IMC-FDU-TZBC-NO in biological systems, the selectivity
2
of IMC-FDU-TZBC-NO towards NTR over other biologically relevant some metal ions, amino
2
acids, and biological reductants was evaluated by determination the changes of fluorescence
intensity. The IMC-FDU-TZBC-NO was allowed to incubate with these bio-related substances
2
o
separately under N atmosphere at 37 C for 24 h, and then the fluorescence intensity of the
2
mixture at λex/em=380/480 nm was recorded. As shown in Figure 2B, IMC-FDU-TZBC-NO was
2
reduced solely by NTR to produce a strong fluorescence. However, it displayed almost no
2-
2-
fluorescence when IMC-FDU-TZBC-NO was mixed with other reductants (Vc, S , and SO ),
2
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biothreitol (Cys and GSH), saccharides (Glu and Fru), amino acids (Asp, Met, and Ala), and metal
+
2+
2+
3+
2+
ions (K , Zn , Fe , Fe , and Cu ). This result indicated that IMC-FDU-TZBC-NO exhibited unique
2
selectivity towards NTR.
Figure 3
Figure 3. (A) The effects of incubation time on the percentage of FDU released from
IMC-FDU-TZBC-NO (100 μM) determined by HPLC method, which was incubated with NTR (10
2
-1
μg mL ) under N atmosphere. (B) The effects of various relevant species (10 μg/mL of NTR, 10
2
+
2+
3+
2+
2+
2-
2
mM of GSH, Cys, Vc, Glu, Fru, Asp, Met, Ala, K , Zn , Fe , Fe , Cu , S , SO ) on the
3
fluorescence intensity of IMC-FDU-TZBC-NO (100 μM) in PBS buffer (containing 1% DMSO, pH
2
7
.4, incubation time: 24 h). (C) The effects of incubation time on the fluorescence intensity of
IMC-FDU-TZBC-NO (100 μM) in serum solution (V /VPBS=9/1, containing 1% DMSO, pH 7.4).
2
serum
(D) The effects of pH values on the fluorescence intensity of IMC-FDU-TZBC-NO (100 μM) and
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IMC-FDU-TZBC-NO incubated with NTR (10 μg/mL) and NADH (2 mM) in PBS buffer (containing
2
1
% DMSO, 10 mM) under N atmosphere for 0.5 h; λex/em= 380/480 nm. The hemolysis rate of
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IMC-FDU-TZBC-NO (E) and FDU-TZBC-NO (F) in fresh blood at different concentrations.
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To assess the stability of IMC-FDU-TZBC-NO in simulant physiological environment, the
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fluorescence changes of IMC-FDU-TZBC-NO in blood serum protein solution (mixture ratio of
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blood serum and PBS buffer is 9 to 1) for different incubating times were measured. As shown in
Figure 3C, nearly no fluorescence and negligible change were observed when the solution was
o
stirred at 37 C for 0, 4, 8, 12, 16, 20, and 24 h. It demonstrated that IMC-FDU-TZBC-NO
2
displayed remarkable stability in blood serum solution.
To further evaluate the effects of pH values on the stability of IMC-FDU-TZBC-NO in PBS
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solution, the fluorescence intensity of IMC-FDU-TZBC-NO solution with various pH values was
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investigated. As shown in Figure 3D, no remarkable fluorescence and changes were observed
when IMC-FDU-TZBC-NO was stirred in PBS solution (In the absence of NTR) over a broad pH
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range of 2-12 for 0.5 h. In the presence of NTR and NADH, IMC-FDU-TZBC-NO exhibited a strong
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green fluorescence in pH range of 4-10. The result revealed that IMC-FDU-TZBC-NO showing
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advantageous stability in PBS solution in pH range of 2-12, and was activated by hypoxia to
release IMC-TZBCM and FDU at pH range of 4 to 10.
To investigate the blood compatibility of IMC-FDU-TZBC-NO2 and FDU-TZBC-NO , the
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hemolysis test in fresh blood was performed. As shown in Figure 3E and 3D, the hemolysis ratio
of IMC-FDU-TZBC-NO and FDU-TZBC-NO (20 mg/mL) was less than 2%, further demonstrating
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the good biocompatibility, superior safe typrofile, and clear potential of IMC-FDU-TZBC-NO2.
Based on above results, the preeminent characteristic of IMC-FDU-TZBC-NO with remarkable
2
stability under simulative physiological environments and excellent selectivity towards hypoxia
endowed IMC-FDU-TZBC-NO with potential application in biological media as a theranostic
2
agent.
Cytotoxicity evaluation. To evaluate the anti-cancer effects of IMC-FDU-TZBC-NO , the
2
viability of HepG-2 or BRL-3A cells co-incubation with IMC-FDU-TZBC-NO under normoxia or
2
hypoxia (3% O ) conditions was determined via MTT assay, using fluorescent dye IMC-TZBCM
2
and active drug FDU as the references. As shown in Figure 4A and 4B, for BRL-3A cells, the cell
viability was decreased a little following the increasing concentration of IMC-TZBCM and
IMC-FDU-TZBC-NO under normoxia conditions, while the viability was decreased significantly
2
with increasing FDU concentration (from 1 to 20 μM). The result indicated that IMC-TZBCM and
IMC-FDU-TZBC-NO showed negligible cytotoxicity to BRL-3A cells under normoxia conditions,
2
while FDU showed high cytotoxicity under same conditions. For HepG-2 cells (as shown in Figure
4
C and 4D), the cell viability was decreased little following the increasing concentration of
IMC-TZBCM under normoxia and hypoxia (3% O ) conditions. Nevertheless, the viability was
2
decreased markedly with increasing FDU concentration under normoxia conditions. When
IMC-FDU-TZBC-NO was co-incubated with HepG-2 cells under normoxia conditions, the viability
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was scarcely changed with increasing IMC-FDU-TZBC-NO concentration from 1 to 20 μM. When
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IMC-FDU-TZBC-NO was co-incubated with HepG-2 cells under hypoxia (3%O ) conditions the cell
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,
viability was decreased obviously with increasing IMC-FDU-TZBC-NO concentration from 1 to 20
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μM. These results illustrated that FDU did not show hypoxia selectivity and displayed high
cytotoxicity under normoxia and hypoxia conditions. However, IMC-FDU-TZBC-NO exhibited
2
high hypoxia selectivity, which displayed high cytotoxicity towards hypoxic cells, and exhibited
non-cytotoxicity towards normal cells.
Figure 4
Figure 4. Cell viability of BRL-3A cells co-incubated with IMC-TZBCM, IMC-FDU-TZBC-NO , or FDU
2
alone under normoxic (O : 20%) conditions for different time (A: 24 h; B: 48 h); Cell viability of
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HepG-2 cells incubated with IMC-TZBCM, IMC-FDU-TZBC-NO , or FDU alone under normoxic (O :
2
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20%) or hypoxic (O : 3%) conditions for different time (C: 24 h; D: 48 h); n=10, data expressed as
2
average ± SE, *, ** and *** means P<0.05, P<0.01 or 0.001, respectively.
Hypoxic Tumor Cell Imaging. Based on the mechanism of release of active drug with
fluorescent dye simultaneously from IMC-FDU-TZBC-NO activated by NTR, we monitored the
2
hypoxia degree of cancer cells and explored the effects of the hypoxia level on the amount of
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released IMC-TZBCM from IMC-FDU-TZBC-NO using laser confocal microscopy. As shown in
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Figure 5A-D, no fluorescence signal was obtained within HepG-2 cells which were co-incubated
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with IMC-FDU-TZBC-NO at 37 C for 12 h under normoxia conditions. However, noticeable
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fluorescence signals were observed within HepG-2 cells which were co-incubated with
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IMC-FDU-TZBC-NO at 37 C for 12 h under various hypoxia conditions (10%, 5%, or 1% O ).
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Moreover, the fluorescence intensity was enhanced with increasing hypoxia degree, and there
was a certain linear relationship between the oxygen content of 20% to 1% (Figure S1). These
results revealed that the rates and amounts of releasing TZ-BCM and FDU were depended on the
hypoxia state within tumor cells, and the degree of hypoxia could be monitored by the
intracellular fluorescence intensity. Thus, the amount of FDU release was dependent on demand
of the individual patient based on the individual hypoxia state.
To evaluate the accumulation of IMC-FDU-TZBC-NO in tumor hypoxia areas, HepG-2 spheres
2
were co-incubated with IMC-FDU-TZBC-NO and the fluorescence signals were measured by laser
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confocal microscopy. As shown in Figure 5E-G, HepG-2 spheres incubated with
IMC-FDU-TZBC-NO exhibited weak fluorescence on the surface and strong fluorescence in the
2
core due to the specific feature of the tumor spheres which was closely related to the tumor
microenvironment (with a normoxic surface and a hypoxic core). However, the weak
fluorescence of Lyso Tracker red in the core and strong fluorescence on the surface was found
due to diffusion of Lyso Tracker red. The result illustrated that the distribution and accumulation
of IMC-FDU-TZBC-NO was extended to the hypoxic tumor microenvironment.
2
Figure 5
Figure 5. Images of the HepG-2 cells co-incubated with IMC-FDU-TZBC-NO under normoxic (A:
2
2
0% O ) and different hypoxic (B: 10% O , C: 5% O , and D: 1% O ) conditions for 12 h. Images of
2 2 2 2
HepG-2 spheres co-incubated with IMC-FDU-TZBC-NO and Lyso Tracker red for 12 h (E). TZ-BCM:
2
λex/em=380/480 nm; Lyso Tracker Red: λex/em=577/590 nm
Targeting effects of IMC-FDU-TZBC-NO2. Considering the superior properties of
IMC-FDU-TZBC-NO , the cell uptake efficiency of IMC-FDU-TZBC-NO dependent on IMC targeting
2
2
function was explored by laser confocal microscopy. After being co-incubated with IMC (500 μM)
for 12 h, the HepG-2 cells and IMC-FDU-TZBC-NO (10 μM) were co-incubated for another 12 h
2
under hypoxia conditions (5% or 1% O ), using HepG-2 cells without co-incubation with IMC as
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the control. As shown in Figure 6, noticeable fluorescence was observed within HepG-2 cells
which were not pretreated with IMC, and the intensity of the fluorescence signal increased with
decreasing the amount of oxygen of incubator. However, the fluorescence intensity was
significant decreased within HepG-2 cells which were pretreated with IMC whether the oxygen
concentration was 1% or 5%, because of IMC has significantly suppressed the expression level of
COX-2 which played a crucial role in tumorigenesis. These results indicated that the introduction
of IMC moiety enhanced the targeting effects of IMC-FDU-TZBC-NO towards cancer cells.
2
Figure 6
Figure 6. Images of the HepG-2 cells co-incubated with IMC-FDU-TZBC-NO in absence of and in
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presence of IMC under hypoxic (A: 1% and B: 5%O ) conditions for 12 h (TZ-BCM: λex/em=380/480
2
nm; Lyso Tracker Red: λex/em=577/590 nm)
Cellular uptake. Subsequently, the cellular uptake efficiency of IMC-FDU-TZBC-NO by HepG-2
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cells under hypoxia (1% O ) conditions was explored. Cultured HepG2 cells were divided into four
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groups. The first group, HepG2 cells were co-incubated with IMC-FDU-TZBC-NO (10 μM) for 12 h
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under hypoxia conditions, and the fluorescence intensity of released TZ-BCM from
IMC-FDU-TZBC-NO was determined by flow cytometer. The second group was co-incubated with
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FDU-TZBC-NO (10 μM) under same conditions. The third group was pretreated with IMC (500
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μM) for 12 h, and then was co-incubated with IMC-FDU-TZBC-NO (10 μM) for 12 h under
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hypoxia conditions. The fourth group without treatment with drug under same conditions was
used as the control. As shown in Figure 7A and 7B, the cellular uptake efficiency of
IMC-FDU-TZBC-NO by HepG-2 cells was higher than that of FDU-TZBC-NO , and much higher
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than that of being pretreated with IMC. These results indicated that the IMC moiety enhanced
the cellular uptake efficiency of IMC-FDU-TZBC-NO by cancer cells.
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Figure 7
Figure 7. The cell uptake efficiency of FDU, FDU-TZBC-NO and IMC-FDU-TZBC-NO by HepG-2
2
,
2
cells under hypoxic (1% O ) conditions, and the cell uptake efficiency of IMC-FDU-TZBC-NO by
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HepG-2 cells treated with IMC under hypoxic (1% O ) conditions (λ =480 nm)
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ex
The solubility characteristic of a prodrug is a key parameter to interpret its bioavailability. So,
the lipid-water partition coefficients of FDU, FDU-TZBC-NO , and IMC-FDU-TZBC-NO were
2
2
measured by the traditional n-octanol–water shake flask method, and the log P values of FDU,
FDU-TZBC-NO , and IMC-FDU-TZBC-NO were calculated as -0.76, 1.67, and 2.21. The result
2
2
suggested that the improved lipophilicity of IMC-FDU-TZBC-NO due to the binding of hypoxic
2
trigger, fluorescent dye precursor, and targeting moiety. In addition, hypoxia targeted drug
delivery is difficult because hypoxic areas in tumors are often poorly vascularized (that's one of
the reasons why it is hypoxic), together with the fact that tumors are in general highly stromal,61
the challenge of FDU with poor tumor penetration due to the low lipophilicity could be
dramatically increased by prodrug of FDU with improved lipophilicity as Wang reported.62
Therefore, the tumor penetration of FDU IMC-FDU-TZBC-NO was improved by its enhanced
2
lipophilicity due to the binding of hypoxic trigger, fluorescent dye precursor, and targeting
moiety.
In vivo antitumor activity. Due to its significant cytotoxicity to hypoxia cancer cells, the in vivo
evaluation of prodrug IMC-FDU-TZBC-NO against xenograft mice vaccinated with HepG-2 cells
2
was investigated. Mice were intravenously tail vein-injected with IMC-FDU-TZBC-NO2,
FDU-TZBC-NO and PBS (control group) at a dose of 10 mg/kg every 3 days with an intermittent
2
q2d*7 schedule, respectively. The tumor volumes of groups that were injected with
FDU-TZBC-NO or IMC-FDU-TZBC-NO were recorded. As shown in Table S1 and Figure 8,
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2
compared with the control, both IMC-FDU-TZBC-NO and FDU-TZBC-NO inhibited HepG-2 tumor
2
2
growth comparing with the control group, showing low toxicity with no magic weight loss during
the treatment process (Figure 8A and 8B). It is worth noting that according to the experimental
results, the tumor inhibition rate of IMC-FDU-TZBC-NO (82.7%) is significantly higher than that
2
of FDU-TZBC-NO (51%), which may be due to the targeting effect of IMC. In addition, the
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photograph of the resected tumours from representative mice intuitively demonstrated that the
tumor sizes of mice injected with IMC-FDU-TZBC-NO group were much smaller than that of
2
injected with FDU-TZBC-NO and PBS (Figure 8C and 8D), which could be attributed to the IMC
2
moiety enhancing the targeting and cellular uptake efficiency of IMC-FDU-TZBC-NO combination
2
with the release of the FDU induced by hypoxia. Meanwhile, to investigate the toxicity of
FDU-TZBC-NO and IMC-FDU-TZBC-NO , mice body weight was measured during the treatment
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period. As shown in Figure 8B, the body weight having no obvious change indicated that
FDU-TZBC-NO and IMC-FDU-TZBC-NO had almost no toxicity in mice produced during the 22
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days treatments. The TUNEL staining exhibited much more destruction within tumor tissue of
mice treated with IMC-FDU-TZBC-NO than that of treated with FDU-TZBC-NO due to the IMC
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moiety improving the efficiency of tumor-targeting and the cellular uptake efficiency of
IMC-FDU-TZBC-NO2 (Figure 8E). These results demonstrated that the IMC-guided and
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Figure 8
Figure 8. The relative tumor volume normalized to initial volume (A), body weight (B), and
average tumor weight changes (C) in 22 days after various treatments (injected with
IMC-FDU-TZBC-NO , FDU-TZBC-NO , or PBS; n= 5, data expressed as average ± SE, *, ** and ***
2
2
means P<0.05, P<0.01 or 0.001, respectively). Representative histological photomicrograph (D) of
HepG-2-cell-inoculated xenograft mice injected with IMC-FDU-TZBC-NO , FDU-TZBC-NO , and
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PBS (Control). TUNEL stained images of cancer tissues from the HepG-2-cell-inoculated xenograft
mice after various treatments for 22 days (E).
Besides, to assess the systemic toxicity of IMC-FDU-TZBC-NO in vivo, a further histological
2
study was conducted on tumor and major organs through hematoxylin and eosin (H&E) staining.
As shown in Figure 9, compared with the groups treated with FDU-TZBC-NO and PBS, the mice
2
treated with IMC-FDU-TZBC-NO exhibited significantly more apoptotic cells in tumor tissue.
2
Moreover, the major organs like the heart, spleen, kidney, liver, and lung of xenograft mice
vaccinated with HepG-2 cells had almost same morphologies to the control group, suggested
that IMC-FDU-TZBC-NO displayed no notable side effect on the major organs of mice. However,
2
starting from the 22th day, tumor-bearing mice in the control group began to die off. Taken
together, IMC-FDU-TZBC-NO could achieve high inhibition effect to tumor growth and no side
2
effect on normal organs, due to the integration of the targeting strategy and prodrug activated
by hypoxia.
Figure 9
Figure 9. H&E staining of the sacrificed cancer tissues and major organs after various treatments
for 22 d
■
CONCLUSION
In conclusion, a new theranostic prodrug IMC-FDU-TZBC-NO2 based on COX-2 inhibitor
IMC-guiding combination hypoxia-activation for targeted chemotherapy, releasing drug
on-demand, combination therapy under the guidance of imaging was developed.
IMC-FDU-TZBC-NO showed a high targeting specificity to tumor cells, a specificand predominant
2
imaging property to hypoxia microenvironment, and excellent stability in physiological solutions.
Furthermore, the IMC moiety improved the cellular uptake efficiency of IMC-FDU-TZBC-NO . In
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addition, the hypoxia response group enhanced the hypoxic selectivity towards hypoxic tumor
cells and reduced the cytotoxicity to normal cells. Eventually, IMC-FDU-TZBC-NO showed
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significant inhibition tumor growth of mice in xenograft mice vaccinated with HepG-2 cells,
exhibited no toxic side effects on heart, liver, spleen, lung and kidney. This strategy will offer a
platform for design a hypoxia-activated and tumor-targeted anticancer theranostic agent for
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targeted chemotherapy, releasing active drug based on hypoxic status, non-invasive and the
real-time monitoring of hypoxia and active drug release, inhibition tumor growth with high
efficiency and no side effects. To improve the diagnostic effect, the future research of our group
should be to improve the strategy of monitoring of hypoxia and active drug release which will be
operated in the near infrared wavelength.
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EXPERIMENTAL SECTION
Materials. 4-Bromo-3-methoxyaniline,
-ethoxycarbonylbenzeneboronic acid,
-dimethylaminopyridine (DMAP),
4-nitrobenzyl
3-bromopropyne,
N,N'-diisopropyl
bromide,
floxuridine
carbodiimide
hydrochloride,
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(FDU),
(DIC),
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HATU),
tetrakis(triphenylphosphine)palladium, 1-amino-3-chloropropane, and IMC were purchased form
Energy Chemical (Shanghai, China). Potassium carbonate, sodium carbonate, sodium sulfate,
copper sulfate pentahydrate, ammonium chloride, sodium dithionate, sodium hydroxide,
petroleum ether (PE), methanol (MeOH), ethyl acetate (EA), ethyl ether, 1,4-dioxane,
tetrahydrofuran (THF), N,N-dimethylformamide (DMF), acetonitrile (ACN), dichloromethane
(DCM), dimethyl sulfoxide (DMSO), concentrated hydrochloric acid, and concentrated sulfuric
acid were all commercially available analytically which were purchased from Tianjin Kemiou
Chemical Reagent Co. Ltd. (Tianjin, China). L-Ascorbic acid (Vc), L-cysteine, L-glutamic acid, and
L-alanine were obtained from Alfa Aesar Chemical Co. Ltd. (Shanghai, China). Nicotinamide
adenine dinucleotide (NADH) and sodium azide (NaN ) were purchased from Sigma-Aldrich Co.
3
LLC (USA). Indomethacin (IMC), trifluoroacetic acid (TFA), benzyl penicillin, streptomycin,
thiazolyl blue tetrazolium bromide (MTT), and NTR (≥100 units/mg) were purchased from Sigma
Chemical Co (USA). Lyso Tracker Red was obtained from Life Technology (New York, USA).
BRL-3A and HepG-2 cell lines were obtained from Beijing Union Cell Resource Center (China).
Immunodeficient BALB/c (nu/nu) mice (four-weeks-old) were achieved from Weitong Lihua
Experimental Animal Technology Co Ltd (Beijing, China).
Instrumentation. NMR was measured on a Bruker AVANCEIII Spectrometer (Germany). MS
was measured on a 4000 QTRAP LC-MS (USA). IR was performed on a spectrophotometer
(Thermo Scientific Nicolet iS10, USA). Fluorescence measurements were performed on a
Fluorescence Spectrophotometer (Hitachi F-7000, Japan). The absorbance was measured on a
Bio-Rad Model 680 microplate spectrophotometer (USA). The relative retention time was
measured on a HPLC system (Agilent 1260, USA), which was equipped with quaternary G1311C
pump, HC-C18 (Agilent, 250 mm × 4.6 mm, 5 μm), and G1314F UV-vis detector. Fluorescence
imaging was measured on an LSM 880 Confocal Microscope (Germany). Cellular uptake was
checked by a FACS-Calibur Flow Cytometer (BD Biosciences, USA).
Synthesis of 4-bromo-N-ethyl-3-methoxyaniline (1). 4-Bromo-3-methoxyaniline (5.01 g, 24.75
mmol), dry DMF (30 mL), bromoethane (3.10 g, 28.46 mmol), and K CO (10.26 g, 74.24 mmol)
2
3
o
were heated to 100 C for 14 h. Followed by cooling and being poured into 50 mL of water, the
2
mixture was extracted with EA (50 mL × 3), washed with saline (50 mL ×3), and dried with Na SO4
sequentially. Then, EA was removed, and compound 1 was isolated as yellow liquid (2.51 g, 44%
1
yield) from the crude by column chromatography isolation (V /V =1/50). H NMR (600 MHz,
EA PE
DMSO-d , ppm): δ 7.16 (d, J = 8.4 Hz, 1 H), 6.27 (d, J = 2.4 Hz, 1 H), 6.09 (dd, J = 9.0, 2.4 Hz, 1 H),
6
1
3
5.74 (s, 1 H), 3.75 (s, 3 H), 3.02 (m, 2 H), 1.15 (t, J = 7.2 Hz, 3 H). C NMR (150 MHz, CDCl , ppm):
3
δ 156.41, 149.16, 133.29, 106.06, 98.16, 97.44, 55.98, 38.58, 14.72. MS (m/z): Calcd. for
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4
5
5
5
5
5
5
5
5
5
5
6
+
C H BrNO, 230.0 [M+H] ; Found, 229.8.
9
12
Synthesis of 2-bromo-5-(ethylamino) phenol (2). Compound 1 (3.02 g, 13.04 mmol), boron
o
tribromide (6.53 g, 26.08 mmol), and dry DCM (20 mL) were stirred for 2 h at 30 C. After being
added 50 mL of water and adjusted to pH 7 with Na CO (aq.), the obtained mixture was
2
3
extracted with DCM (50 mL × 3), and dried with MgSO . Then, DCM wasremoved, and compound
4
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 was generated as white solid (2.23 g, 79% yield) from the crude by column chromatography
1
isolation (V /V =1/30). Mp: 95.7-96.2 °C. H NMR (600 MHz, DMSO-d , ppm): δ 9.63 (s, 1 H),
EA PE
6
7
.06 (d, J = 8.4 Hz, 1 H), 6.17 (d, J = 2.4 Hz, 1 H), 5.98 (dd, J = 8.6, 2.4 Hz, 1 H), 5.59 (t, J = 7.2 Hz, 1
H), 2.93 (m, 2 H), 1.13 (t, J = 7.2 Hz, 3 H). 13C NMR (150 MHz, CDCl , ppm): δ 152.90, 149.56,
3
+
131.89, 107.34, 99.61, 96.79, 38.47, 14.67. MS (m/z): Calcd. for C H BrNO, 216.0 [M+H] ; Found,
8
10
2
15.9.
Synthesis of t-butyl (4-bromo-3-hydroxyphenyl)ethyl carbamate (3). Boc anhydride (3.94 g,
18.05 mmol), 1,4-dioxane (20 mL), and compound 2 (3.00 g, 13.88 mmol) were stirred for 24 h at
o
3
5 C. After drying, compound 3 was generated as white solid (3.07 g, 70% yield) from the
1
residue by column chromatography isolation (V /V =1/30). Mp: 106.0-106.6 °C. H NMR (600
EA PE
MHz, CDCl , ppm): δ 7.39 (d, J = 8.4 Hz, 1 H), 6.88 (s, 1 H), 6.68 (d, J = 8.4 Hz, 1 H), 5.57 (s, 1 H),
3
13
3
1
.64 (q, J = 7.2 Hz, 2 H), 1.44 (s, 9 H), 1.14 (t, J = 7.2 Hz, 3 H). C NMR (150 MHz, CDCl , ppm): δ
3
54.40, 152.66, 143.14, 131.86, 120.38, 114.94, 107.30, 80.57, 44.95, 28.33, 13.79. MS (m/z):
+
Calcd. for C H BrNO 338.1 [M+Na] ; Found, 338.2.
13 18
3,
Synthesis of t-butyl (4-bromo-3-((4-nitrobenzyl)oxy)phenyl)ethyl carbamate (4). Compound 3
(
5.00 g, 15.81 mmol), 4-nitrobenzyl bromide (5.12 g, 23.72 mmol), dry ACN (20 mL), and K CO3
2
(
6.57 g, 47.46 mmol) were refluxed for 3 h. After ACN being removed, compound 4 was
generated as yellow solid (5.69 g, 80% yield) from the residue by column chromatography
1
isolation (V /V =1/20). Mp: 127.4-128.2 °C. H NMR (600 MHz, CDCl , ppm): δ 8.27 (d, J = 8.4
EA PE
3
Hz, 2 H), 7.69 (d, J = 8.4 Hz, 2 H), 7.53 (d, J = 8.4 Hz, 1 H), 6.83 (s, 1 H), 6.75 (d, J = 8.4 Hz, 1 H),
5.22 (s, 2 H), 3.63 (q, J = 7.2 Hz, 2 H), 1.43 (s, 9 H), 1.12 (t, J = 7.2 Hz, 3 H). 13C NMR (150
MHz,CDCl , ppm): δ 154.41, 154.10, 147.68, 143.69, 143.02, 133.21, 127.45, 123.86, 121.05,
3
1
13.11, 109.45, 80.49, 69.65, 44.97, 28.35, 13.89.
Synthesis of ethyl 4'-((t-butoxycarbonyl)ethylamino)-2'-((4-nitrobenzyl)oxy)-1,1'-
biphenyl-2-carboxylate (5). Tetrakis(triphenylphosphine)palladium (1.01 mg, 0.87 mmol),
compound 4 (2.40 g, 5.33 mmol), 2-ethoxycarbonylbenzeneboronic acid (1.25 g, 6.40 mmol),
o
NaHCO (1.34 g, 16.00 mmol), and 25 mL of H O/DMF (1/4) were heated to 120 C and stirred for
3
2
4 h under N . After solvent being removed, compound 5 was isolated as yellow solid (1.86 g, 67%
2
yield) from the residue by column chromatography isolation (V /V =1/15). Mp: 113.5-114.1 °C.
EA PE
1
H NMR (600 MHz,CDCl , ppm): δ 8.13 (d, J = 7.8 Hz, 2 H), 7.94 (d, J = 7.8 Hz, 1 H), 7.56 (t, J = 7.2
3
Hz, 1 H), 7.45 (t, J = 7.2 Hz, 1 H) 7.33 (d, J = 8.4 Hz, 3 H), 7.23 (d, J = 8.4 Hz, 1 H), 6.93 (d, J = 7.8
Hz, 1 H), 6.82 (s, 1 H), 5.05 (s, 2 H ), 4.04 (s, 2 H), 3.69 (q, J = 7.2 Hz, 2 H), 1.46 (s, 9 H), 1.18 (t, J =
1
3
7
1
.2 Hz, 3 H), 0.98 (t, J = 7.2 Hz, 3 H). C NMR (150 MHz, CDCl , ppm): δ 167.94, 154.79, 154.36,
3
47.35, 144.26, 143.05, 138.29, 131.82, 131.51, 131.38, 129.99, 129.71, 129.56, 127.40, 127.13,
123.54, 119.80, 111.70, 80.20, 69.42, 60.57, 45.17, 28.39, 13.98, 13.76. MS (m/z): Calcd. for
+
C H N O , 543.2 [M+Na] ; Found, 542.4.
2
9 32 2 7
Synthesis of ethyl 4'-(ethylamino)-2'-((4-nitrobenzyl)oxy)-1,1'-biphenyl-2-carboxylate (6).
Compound 5 (3.00 g, 5.77 mmol), hydrochloric acid (10 mL), and 1,4-dioxane (20 mL) were
o
stirred for 4 h at 35 C. After being dispersed into 100 mL of water and adjusted to pH 8 with
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7
8
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Na CO (aq.), the resulting solution was extracted with DCM (50 mL × 3), washed with saline (50
2
3
mL × 3), and dried with MgSO sequentially. Then, DCM was removed, and compound 6 was
4
obtained as orange solid (1.87 g, 77% yield) from the residue by column chromatography
1
isolation (V /V =1/10). Mp: 135.1-135.9 °C. H NMR (600 MHz, DMSO-d , ppm): δ 8.15 (d, J =
EA PE
6
9
.0 Hz, 2 H), 7.69 (d, J = 7.8 Hz, 1 H), 7.55 (t, J = 7.8 Hz, 1 H), 7.48 (d, J = 8.4 Hz, 2 H), 7.37 (t, J =
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
7.8 Hz, 1 H), 7.31 (d, J = 7.2 Hz, 1 H), 6.91 (d, J = 9 Hz, 1 H), 6.24 (d, J = 7.2 Hz, 2 H), 5.65 (t, J = 5.4
Hz, 1 H), 5.10 (s, 2 H), 3.97 (q, J = 7.2 Hz, 2 H), 3.05 (m, 2 H), 1.14 (t, J = 7.2 Hz, 3 H), 0.94 (t, J = 7.2
1
3
Hz, 3 H). C NMR (150 MHz, DMSO-d , ppm): δ 167.86, 155.33, 149.97, 146.73, 145.33, 138.80,
6
1
31.90, 131.31, 131.15, 130.42, 128.75, 127.50, 126.13, 123.23, 117.87, 104.71, 96.82, 68.20,
+
59.99, 37.33, 14.35, 13.60. MS (m/z): Calcd. for C H N O , 421.2 [M+H] ; Found, 421.4.
2
4 24 2 5
Synthesis
of
ethyl
4'-(ethyl(prop-2-yn-1-yl)amino)-2'-((4-nitrobenzyl)oxy)-1,1'-
biphenyl-2-carboxylate (7). Compound 6 (2.00 g, 4.76 mmol), propargyl bromide (0.85 g, 7.15
o
mmol), dry ACN (20 mL), and 1.97 g of K CO (14.28 mmol) were stirred for 12 h at 60 C. Then,
2
3
K CO and ACN were removed sequentially, and compound 7 was generated as yellow-brown
2
3
solid (873 mg, 40% yield) from the residue by column chromatography isolation (V /V =1/20).
EA PE
1
Mp: 104.8-105.7 °C. H NMR (600 MHz, DMSO-d , ppm): δ 8.15 (d, J = 8.4 Hz, 2 H), 7.73 (d, J = 7.8
6
Hz, 1 H), 7.57 (t, J = 7.2 Hz, 1 H), 7.48 (d, J = 8.4 Hz, 2 H), 7.40 (t, J = 7.2 Hz, 1 H), 7.34 (d, J = 7.8
Hz, 1 H), 7.03 (d, J = 8.4 Hz, 1 H), 6.47 (d, J = 8.4 Hz, 1 H), 6.44 (s, 1 H), 5.17 (s, 2 H), 4.10 (s, 2 H),
3
.97 (q, J = 7.2 Hz, 2 H), 3.42 (q, J = 7.2 Hz, 2 H), 3.06 (s, 1 H), 1.10 (t, J = 7.2 Hz, 3 H), 0.92 (t, J =
1
3
7.2 Hz, 3 H). C NMR (150 MHz, CDCl , ppm): δ 168.39, 155.69, 148.67, 147.29, 144.87, 138.94,
3
1
32.00, 131.55, 131.31, 130.77, 129.53, 127.22, 126.66, 123.53, 121.13, 107.05, 99.34, 71.79,
+
6
9.51, 65.56, 60.51, 45.85, 39.76, 13.86, 12.47. MS (m/z): Calcd. for C H N O , 459.2 [M+H] ;
2
7 26 2 5
Found, 459.7.
Synthesis
of
4'-(ethyl(prop-2-yn-1-yl)amino)-2'-((4-nitrobenzyl)oxy)-1,1'-biphenyl-2-
carboxylic acid (8). Compound 7 (500 mg, 1.09 mmol) dissolved in 4 mL of 1,4-dioxane and 30%
o
sulfuric acid (20 mL) were heated at 100 C for 12 h. Then, the mixture was cooled down,
dispersed into 50 mL of water, adjusted to pH 6 with Na CO (aq), extracted with DCM (50 mL ×
2
3
3
), washed with saline (50 mL ×3), and dried with MgSO sequentially. After DCM being removed,
4
compound 8 was afforded as yellow solid (235 mg, 50 % yield) from residue by column
1
chromatography isolation (VMeOH/VDCM=1/80). Mp: 191.5-192.2 °C. H NMR (600 MHz, DMSO-d ,
6
ppm): δ 12.35 ( s, 1 H), 8.15 (d, J = 8.4 Hz, 2 H), 7.74 (d, J = 7.8 Hz, 1 H), 7.55 (d, J = 9 Hz, 2 H), 7.52
(d, J = 7.2 Hz, 1 H), 7.37 (t, J = 7.8 Hz, 1 H), 7.30 (d, J = 7.2 Hz, 1 H), 7.02 (d, J = 8.4 Hz, 1 H), 6.44
(dd, J = 8.4 Hz, J = 2.4 Hz, 1 H), 6.38 (d, J = 1.8 Hz, 1 H), 5.19 (s, 2 H), 4.08 (d, J = 1.8 Hz, 2 H),
1
2
13
3
.39 (q, J = 7.2 Hz, 2 H), 3.07 (s, 1 H), 1.08 (t, J = 7.2 Hz, 3 H). C NMR (150 MHz, DMSO-d , ppm):
6
δ 169.02, 155.55, 148.09, 146.71, 145.50, 138.29, 132.87, 131.42, 130.75, 130.42, 128.86,
127.71, 126.29, 123.29, 119.55, 105.70, 98.38, 81.03, 73.99, 68.64, 44.84, 38.85, 12.13. MS
+
(
m/z): Calcd. for C H N O , 431.2 [M+H] ; Found, 431.3.
25 22 2 5
Synthesis of FDU-TZBC-NO . Compound 8 (81 mg, 0.19 mmol), dry DMF (0.5 mL), DMAP (10
2
o
mg, 0.07 mmol), DIC (94 mg, 0.74 mmol), and FDU (46 mg, 0.19 mmol) were heated to 50 C and
stirred for 24 h. After being cooled down and dispersed into 5 mL of water, the obtained mixture
was extracted with DCM (5 mL ×4), washed with saline (5 mL×4), and dried with MgSO4
sequentially. Then, DCM was removed, and FDU-TZBC-NO was afforded as yellow-green solid
2
(22 mg, 18% yield) from the residue by column chromatography isolation (VMeOH/VDCM=1/50).
1
Mp: 91.5-92.2 °C. H NMR (600 MHz, DMSO-d , ppm): δ 11.78 (s, 1 H), 8.11 (d, J = 9 Hz, 2 H), 7.75
6
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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
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5
5
5
5
5
5
5
6
(
d, J = 7.8 Hz, 1 H), 7.65 (d, J = 6.6 Hz, 1 H), 7.59 (t, J = 7.2 Hz, 1 H), 7.45 (d, J = 9 Hz, 2 H), 7.40 (t, J
=
7.8 Hz, 1 H), 7.35 (d, J = 7.8 Hz, 1 H), 7.05 (d, J = 8.4 Hz, 1 H), 6.47 (dd, J = 8.4 Hz, J = 2.4 Hz, 1
1 2
H), 6.42 (s, 1 H), 6.06 (t, J = 7.2 Hz, 1 H), 5.31 (d, J = 4.8 Hz, 1 H), 5.16 (s, 2 H), 4.21 (s, 2 H), 4.10 (s,
2
1
H), 3.99 (m, 1 H), 3.79 (q, J = 4.8 Hz, 1 H), 3.42 (q, J = 7.2 Hz, 2 H), 3.07 (s, 1 H), 2.01 (m, 2 H),
.10 (t, J = 7.2 Hz, 3 H). 13C NMR (150 MHz, DMSO-d , ppm): δ 167.47, 156.79, 155.27, 148.81,
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
148.41, 146.68, 145.18, 140.61, 139.08, 138.36, 131.61, 131.48, 131.12, 130.37, 128.93, 127.51,
1
4
26.48, 124.28, 123.24, 118.80, 106.00, 98.34, 84.37, 83.74, 80.96, 74.00, 70.09, 68.44, 64.33,
+
4.87, 38.94, 12.08. MS (m/z): Calcd. for C H FN O , 687.2 [M+K] ; Found, 697.8.
3
4
31
4 9
Synthesis of IMC-N . 1-Amino-3-chloropropane hydrochloride (100 mg, 0.77 mmol), NaN (50
3
3
mg, 0.77 mmol), and water (3 mL) were refluxed for 16 h. Then, the solution was cooled down,
neutralized with NaOH (aq), extracted with Et O (2 mL × 3), and dried with K CO . Fellowed by
2
2
3
Et O being evaporated, NH CH CH CH N was obtained aslight yellow oily liquid (69 mg, 90%
2
2
2
2
2 3
yield).
IMC (500 mg, 1.40 mmol), HATU (638 mg, 1.68 mmol), and dry DMF (10 mL) were stirred at
o
3
5 C for 1 h. Followed by adding NH CH CH CH N (127 mg, 1.27 mmol), the solution was stirred
2 2 2 2 3
for another 1 h. After being dispersed into 100 mL of water, the obtained mixture was extracted
with EA (50 mL ×3), washed with saline (50 mL ×3), and dried with MgSO sequentially. Fellowed
4
by EA being removed, IMC-N was obtained as light green solid (223 mg, 40% yield) from the
3
1
crude by column chromatography isolation (Eluent:V /V =1/5). Mp: 159.3-160.5 °C. H NMR
EA PE
(600 MHz, CDCl , ppm): δ 7.64 (d, J = 8.4 Hz, 2 H), 7.48 (d, J = 8.4 Hz, 2 H), 6.89 (d, J = 2.4 Hz, 1 H),
3
6
.85 (d, J = 9.0 Hz, 1 H), 6.70 (dd, J = 9.0 Hz, J = 2.4 Hz, 1 H), 5.89 (s, 1 H), 3.82 (s, 3 H), 3.64 (s, 2
1 2
1
3
H), 3.29 (q, J = 6.6 Hz, 2 H), 3.26 (t, J = 6.6 Hz, 2 H), 2.39 (s, 3 H), 1.70 (m, 2 H). C NMR (150 MHz,
CDCl , ppm): δ 170.07, 168.33, 156.33, 139.61, 136.42, 133.56, 131.18, 130.93, 129.24, 128.84,
3
1
15.15, 112.74, 112.28, 100.83, 55.77, 49.47, 37.46, 32.22, 28.60, 13.22. MS (m/z): Calcd. for
+
C H ClN O , 461.2 [M+Na] ; Found, 462.1.
2
3
23
4 3
Synthesis of IMC-TZBC-NO . Compound 8 (215 mg, 0.5 mmol), IMC-N (220 mg, 0.5 mmol), 6
2
3
mL of THF, and CuSO •5H O (71 mg, 0.28 mmol) dissolved in water (3 mL) were stirred at rt for 5
4
2
min. Followed by adding 3 mL of Vc (50 mg, 0.28 mmol) solution, the mixture was heated to 55
o
C and stirred for 10 h under N . After being cooled down, the resulting mixture was dispersed
2
into 100 mL of NH Cl (aq), extracted by EA (80 mL × 3), washed twice with 50 mL of saline, and
4
dried with MgSO sequentially. Fellowed by EA being removed, IMC-TZBC-NO was afforded as
4
2
yellow-brown solid (243 mg, 70% yield) from the residue by column chromatography isolation
1
(VMeOH/VDCM=1/50). Mp: 215.2-215.8 °C. H NMR (600 MHz, DMSO-d , ppm): δ 12.29 (s, 1 H), 8.12
6
(d, J = 8.4 Hz, 2 H), 8.09 (d, J = 5.4 Hz, 1 H), 7.90 (s, 1 H), 7.72 (d, J = 7.2 Hz, 1 H), 7.67 (d, J = 8.4
Hz, 2 H), 7.63 (d, J = 8.4 Hz, 2 H), 7.51 (d, J = 9 Hz, 2 H), 7.49 (d, J = 7.2 Hz, 1 H), 7.35 (t, J = 7.8 Hz,
1 H), 7.26 (d, J = 7.8 Hz, 1 H), 7.10 (d, J = 2.4 Hz, 1 H), 6.97 (d, J = 8.4 Hz, 1 H), 6.93 (d, J = 9 Hz, 1
H), 6.69 (dd, J = 9 Hz, 2.4 Hz, 1 H), 6.42 (dd, J = 8.4, 1.8 Hz, 1 H), 6.36 (s, 1 H), 5.14 (s, 2 H), 4.49 (s,
2
2
H), 4.30 (t, J = 7.2 Hz, 2 H), 3.72 (s, 3 H), 3.50 (s, 2 H), 3.41 (q, J = 7.2 Hz, 2 H), 3.05 (q, J = 6.6 Hz,
1
3
H), 2.21 (s, 3 H), 1.92 (m, 2 H), 1.06 (t, J = 7.2 Hz, 3 H). C NMR (150 MHz, DMSO-d , ppm): δ
6
169.56, 169.27, 167.80, 155.54, 155.47, 148.40, 146.66, 145.52, 144.69, 138.35, 137.54, 135.13,
1
1
34.21, 132.76, 131.37, 131.09, 130.83, 130.74, 130.53, 130.28, 128.99, 128.85, 127.64, 126.15,
23.24, 122.73, 118.50, 114.53, 114.21, 111.22, 104.77, 101.78, 97.32, 68.40, 55.38, 47.09,
+
44.90, 44.44, 35.99, 31.14, 29.94, 13.31, 12.06. MS (m/z): Calcd. for C H ClN O , 892.3 [M+Na] ;
4
7
44
7 8
Found, 893.0.
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Synthesis of IMC-FDU-TZBC-NO . IMC-TZBC-NO (102 mg, 0.11 mmol), DMAP (6 mg, 0.05
2
2
mmol), DIC (54 mg, 0.43 mmol), FDU (28 mg, 0.11 mmol), and dry DMF (1 mL) were heated to 50
oC and stirred for 24 h. After cooling and adding 5 mL of water, the obtained mixture was
extracted with DCM (5 mL × 3), washed with saline (5 mL× 2), and dried with MgSO . Then, DCM
4
was removed, IMC-FDU-TZBC-NO was afforded as yellow-green solid (20 mg, 16% yield) from
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
the crude by column chromatography isolation (Eluent: VMeOH/VDCM=1/40). Mp: 130.9-132.4 °C.
1
H NMR (600 MHz, DMSO-d , ppm): δ 11.79 (s, 1 H), 8.14 (t, J = 6 Hz, 1 H), 8.08 (d, J = 9.0 Hz, 2 H),
6
7
7
.91 (s, 1 H), 7.73 (d, J = 7.2 Hz, 1 H), 7.68 (d, J = 8.4 Hz, 2 H), 7.63 (d, J = 8.4 Hz, 3 H), 7.57 (t, J =
.2 Hz, 1 H), 7.42 (d, J = 9.0 Hz, 2 H), 7.38 (t, J = 7.2 Hz, 1 H), 7.31 (d, J = 7.8 Hz, 1 H), 7.11 (d, J =
2.4 Hz, 1 H), 6.99 (d, J = 7.2 Hz, 1 H), 6.93 (d, J = 9 Hz, 1 H), 6.69 (dd, J = 7.2 Hz, J = 2.4 Hz, 1 H),
1
2
6
2
.43 (dd, J = 8.4, 1.8 Hz, 1 H), 6.38 (s, 1 H), 6.06 (t, J = 6.6 Hz, 1 H), 5.34 (d, J = 4.2 Hz, 1 H), 5.12 (s,
H), 4.52 (s, 2 H), 4.30 (t, J = 7.2 Hz, 2 H), 4.19 (m, 2 H), 3.93 (m, 1 H), 3.79 (q, J = 4.2 Hz, 1 H),
3.72 (s, 3 H), 3.50 (s, 2 H), 3.43 (q, J = 7.2 Hz, 2 H), 3.17 (d, J = 5.4 Hz, 1 H), 3.05 (q, J = 6.6 Hz, 2 H),
13
2
1
.21 (s, 3 H), 1.92 (m, 3 H), 1.07 (t, J = 7.2 Hz, 3 H). C NMR (150 MHz, DMSO-d , ppm): δ 167.80,
6
67.60, 155.53, 155.33, 148.80, 148.63, 146.59, 145.18, 144.67, 140.57, 139.03, 138.33, 137.53,
135.09, 134.17, 131.54, 131.37, 131.10, 131.07, 130.82, 130.24, 128.97, 127.44, 126.31, 123.17,
1
5
22.74, 117.74, 114.50, 114.26, 111.25, 101.72, 97.18, 84.36, 83.79, 70.10, 68.34, 64.29, 60.32,
5.33, 48.54, 47.10, 44.98, 44.47, 35.96, 34.62, 31.08, 29.90, 18.57, 13.77, 13.28, 11.98.
Synthesis of IMC-TZBCM. 4 mL of Na S O (200 mg, 1.15 mmol) solution, IMC-TZBC-NO (200
2
2
4
2
mg, 0.23 mmol), DCM (2 mL), and MeOH (10 mL) were stirred for 2 h at rt. After being dispersed
into 50 mL of water, the resulting mixture was extracted with DCM (40 mL ×4), washed with
saline (40 mL × 3), and dried with MgSO sequentially. Then, DCM was removed, IMC-TZBCM was
4
generated as yellow green solid (138 mg, 84% yield) from the crude by column chromatography
1
isolation (VMeOH/VDCM=1/100). Mp: 169.2-170.1 °C. H NMR (600 MHz, DMSO-d , ppm): δ 8.60 (d,
6
J = 7.8 Hz, 1 H), 8.13 (d, J = 7.8 Hz, 1 H), 8.08 (t, J = 5.4 Hz, 1 H), 8.05 (d, J = 9.0 Hz, 1 H), 7.99 (s, 1
H), 7.82 (t, J = 7.8 Hz, 1 H), 7.67 (d, J = 8.4 Hz, 2 H), 7.63 (d, J = 8.4 Hz, 2 H), 7.47 (t, J = 7.8 Hz, 1
H), 7.09 (d, J = 2.4 Hz, 1 H), 6.93 (d, J = 9 Hz, 1 H), 6.85 (dd, J = 9, 2.4 Hz, 1 H), 6.71 (s, 1 H), 6.68
(
dd, J = 9, 2.4 Hz, 1 H), 4.63 (s, 2 H), 4.31 (t, J = 7.2 Hz, 2 H), 3.72 (s, 3 H), 3.56 (q, J = 7.2 Hz, 2 H),
1
3
3
.48 (s, 2 H), 3.05 (q, J = 6.6 Hz, 2 H), 2.21 (s, 3 H), 1.93 (m, 2 H), 1.16 (t, J = 7.2 Hz, 3 H). C NMR
(150 MHz, DMSO-d , ppm): δ 174.75, 173.05, 166.13, 160.78, 159.48, 157.80, 154.88, 149.32,
6
1
1
42.77, 140.83, 140.35, 139.46, 136.34, 136.06, 135.52, 134.23, 128.20, 128.06, 126.33, 123.55,
19.75, 119.47, 116.48, 114.78, 111.09, 107.06, 106.98, 103.74, 92.33, 60.62, 52.41, 50.08,
+
49.95, 41.24, 36.38, 35.18, 18.56, 17.29. MS (m/z): Calcd. for C H ClN O , 717.3 [M+H] ; Found,
4
0
37
6 5
7
17.0.
Fluorescence measurements of IMC-FDU-TZBC-NO2 co-incubated with NTR.
IMC-FDU-TZBC-NO solution (100 μM) was prepared by dissolving IMC-FDU-TZBC-NO in PBS
A
2
2
buffer (containing 0.1% DMSO, pH=7.4). Then, the IMC-FDU-TZBC-NO solution and 10 μg/mL
2
NTR solution (with 2 mM NADH) were stirred for different time ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
o
1
3, 11, 12, 13, 14, 15, 16, 17, and 18 min) at 37 C under hypoxia conditions. Fluorescence
measurements were performed in a 2.5 × 10 mm quartz cell on a luminescence
spectrophotometer (λ =380 nm, emission was collected at 400-700 nm).
ex
Drug release. FDU solution (1 mM) was diluted by PBS buffer solution (pH=7.4) into 25, 50, 75,
and 100 μM. The standard curve between concentration and peak area was detected through
HPLC.
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Then, 100 μM IMC-FDU-TZBC-NO solution was mixed with 10 μg/mL NTR solution (with 2
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o
mM NADH) and stirred at 37 C under hypoxia conditions for different time (0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 min). Then, the FDU concentrations were detected
through HPLC based on concentration-peak area standard curve.
The chemical composition of IMC-FDU-TZBC-NO reacted with Na S O . Na S O (40 mg, 0.23
2
2
2
4
2 2 4
0
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3
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0
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mmol) and 100 mL of IMC-FDU-TZBC-NO solution (200 μM) were stirred for 0.5 h. Then, the
2
composition of the solution was detected by comparison of HPLC retention times of IMC-TZBCM,
FDU, and IMC-FDU-TZBC-NO (at 254 nm).
2
The analysis was performed using a gradient elution beginning with 5/95 (V/V) water
(containing 0.1% TFA)/CAN, which was reaching 50/50 water (containing 0.1% TFA)/ACN within 7
min, and then to 60/40 water (containing 0.1% TFA)/ACN within 15-20 min, and finally to 100/0
water (containing 0.1% TFA)/ACN within 20-30 min. Flow ratewas 1 mL/min.
Selectivity of IMC-FDU-TZBC-NO . 10 mL of IMC-FDU-TZBC-NO solution (200 μM) was mixed
2
2
with 10 mL of 20 μg/mL NTR solution (with 2 mM NADH) and stirred for 24 h. Similarly, 10 mL of
IMC-FDU-TZBC-NO solution (200 μM) was mixed with 10 mL of 20 mM glutathione, cysteine,
2
ascorbic acid, glutamate, fructose, aspartic acid, methionine, alanine, CuCl , KCl, FeCl , FeCl ,
2
3
2
ZnCl , Na SO , or Na S, respectively. All fluorescence measurements were performed on a
2
2
3
2
fluorescence spectrophotometer (λ =380 nm, collected from 400 to 700 nm, 5/5 nm slit width).
ex
Stability of IMC-FDU-TZBC-NO in blood serum. IMC-FDU-TZBC-NO solution (1 mmol/L) was
2
2
diluted into 200 μM with DMSO/PBS solution (pH=7.4, DMSO/PBS=1/999). Then, the
IMC-FDU-TZBC-NO solution (200 μM) was diluted into 100 μM with 10% of fetal bovine serum.
2
o
Protected from light at 37 C, the fluorescence intensity of IMC-FDU-TZBC-NO solution was
2
measured at 0, 4, 8, 12, 16, 20, and 24 h by fluorescence spectrophotometer (λex/em=380/480
nm).
Influence of pH on stability of IMC-FDU-TZBC-NO . IMC-FDU-TZBC-NO solution (1 mM) were
2
2
diluted into 100 μM by PBS buffer solutions with different pH (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12)
and stirred at 37°C for 0.5 h. The fluorescence intensity was determined.
Next, the IMC-FDU-TZBC-NO solutions in different pH (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) were
2
o
mixed with 10 μg/mL NTR solution (with 2 mM NADH) and stirred at 37 C under hypoxia
conditions for 0.5 h. The fluorescence intensity was determined.
4
Cytotoxicity. HepG-2 or BRL-3A cells (2 × 10 cells/mL) seeded in 96 well plates were incubated
o
in normal oxygen levels at 37 C for 12 h. Subsequently, the cells were co-incubated with
IMC-TZBCM FDU, or IMC-FDU-TZBC-NO solution (1, 5, 10, 15, or 20 μM) under hypoxia (1% O )
,
2
2
or normoxia conditions for another 12 h, respectively. Then, the cells were incubated under
normoxic conditions for 12 or 36 h. Subsequently, the cells were co-incubated with 20 mL of MTT
(5 mg/mL) for another 4 h. At last, the supernatant was dissolved with 100 μL of DMSO, and the
absorbance was determined at 570 nm by a microplate reader. Wells containing culture medium
without cells were used as blanks.
The cultivation of tumor spheres. The HepG-2 spheres were cultured according to reported
method.60
4
Confocal microscopy imaging. HepG-2 cells (2 × 10 cells/mL) seeded in 96 well plates were
incubated in normal oxygen levels at 37 oC for 12 h. Subsequently, the cells and
IMC-FDU-TZBC-NO (10 μM) were co-incubated under normoxia or different hypoxia (10% O , 5%
2
2
o
O , or 1% O ) conditions at 37 C for 12 h. Next, the cells were labeled with Lyso-Tracker Red (30
2
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μg/mL) for 0.5 h and rinsed with PBS buffer twice to remove the medium. Fluorescence imaging
was performed at λ =405 nm.
ex
The co-incubation and imaging of HepG-2 spheres with IMC-FDU-TZBC-NO (10 μM) and
2
Lyso-Tracker Red (30 μg/mL) were performed as the same method.
HepG-2 cells were pretreated in DMEM for 12 h, and washed with PBS buffer (×3). To test the
inhibition effect of IMC, HepG-2 cells were incubated with IMC (500 μM) for 12 h and washed
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with PBS buffer (×3). Then, the HepG-2 cells were incubated with IMC-FDU-TZBC-NO (10 μM) in
2
o
fresh culture medium under different hypoxia (1% O or 5% O ) conditionsat 37 C for 12 h. Next,
2
2
the cells were labeled with Lyso-Tracker Red (30 μg/mL). After being treated 0.5 h, the cells were
rinsed twice with PBS buffer to remove the medium. Fluorescence imaging was performed at
λex= 405 nm.
5
Cellular uptake. HepG-2 cells (1×10 cells per well) were seeded in 6-well plates for 24 h. Then,
HepG-2 cells in a 6-well plate were divided into four groups. The first group, HepG-2 cells and
FDU-TZBC-NO (10 μM) were co-incubated for 12 h. The second group, HepG-2 cells and
2
IMC-FDU-TZBC-NO (10 μM) were co-incubated for 12 h. The third group, HepG-2 cells were
2
pre-incubated with IMC (500 μM) for 12 h, and then were co-incubated with IMC-FDU-TZBC-NO2
(
10 μM) for another 12 h. The fourth group used as the control was treated with PBS. All four
o
groups were cultured under the same conditions (1% O or 5% O ) at 37 C. Single cell
2
2
suspensions were serially prepared by trypsin digestion and washed with PBS. Afterwards, cells
were analyzed by FACS-Calibur flow cytometer.
Hemolysis assessment. Fresh blood (2 mL) of BALB/c (nu/nu) mouse eyeballs was added 4 mL
normal saline and centrifuged to discard the supernatant. The collected red blood cells was
washed with normal saline until the supernatant was clear and suspended into normal saline
solution assigned to 10%. 0.2 mL of red cell suspension was mixed with 0.8 mL
IMC-FDU-TZBC-NO with different concentrations (1, 5, 10, 15, and 20 mg/mL), 0.8 mL water as
2
positive control and 0.8 mL normal saline as negative control. Each group was divided into 3
parallel groups, incubated at 37 oC for 30 min and the supernatant was obtained by
çentrifugation. Samples were taken from 100 μL to 96 well plates, and the absorbance was
measured at 545 nm.
In vivo antitumor activity. Animal experiments were conducted according to the guidelines of
the Hebei University Animal Welfare and Ethical Committee. Balb/c (nu/nu) mice (about 20 g, 4
o
weeks old) were fed in animal room (temperature: 20±2 C; relative humidity: 60±10%;
7
illumination time: 12 h) with free access to water and food. HepG-2 cells (around 1×10 cells
each mouse) were injected into the right flank of the nude mice, all of which developed tumors
3
in 7 d with sizes of about 50 mm by average. These mice were assigned into 3 groups (5
mice/group) randomly, and treated by IMC-FDU-TZBC-NO , FDU-TZBC-NO and PBS (control
2
2
group) via tail vein injection (10 mg/kg) every 3 d, respectively. Tumor volume was recorded. All
mice were sacrificed 22 d after the first treatment, the tumors and major organs (heart, liver,
spleen, lung, and kidney) were excised, weighed and fixed with formalin for paraffin embedding.
Histological examination of tumor tissues. The excised tumors (or Heart, liver, spleen, lung,
and kidney) were fixed using 4% formalin, embedded in paraffin and sectioned into 5 um thick
slices. The sections were stained with H&E for histological examinations and observed under
inverted microscope.
Notes
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The authors declare no competing financial interests.
■
ACKNOWLEDGMENTS
This work was supported by the Priority Strategy project of Key Laboratory of Medicinal
Chemistry and Molecular Diagnosis of Ministry of Education (No. ts2019001), the Natural Science
Foundation of Hebei Province (No. B2019201444, B2019201333), and the Postgraduate’s
Innovation Fund Project of Hebei University (hbu2019ss008).
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