Enhanced Efficacy of Gefitinib in Drug-Sensitive and Drug-Resistant Cancer Cell Lines after Arming with a Singlet Oxygen Releasing Moiety

Article abstract of DOI:10.1002/cmdc.202000036

Attractive results have been achieved with small-molecule target-based drugs in the anticancer field; however, enhancing their treatment effect and solving the problem of drug resistance remain key concerns worldwide. Inspired by the specific affinity of

Full text of DOI:10.1002/cmdc.202000036

Accepted Article
Title: Enhanced Efficacy of Gefitinib in Drug-sensitive and Drug-
resistant Cancer Cell Lines after Arming with a Singlet Oxygen
Releasing Moiety
Authors: Kunshan Huang, Beibei Huo, Dongyao Li, Jinping Xue, and
Juanjuan Chen
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Enhanced Efficacy of Gefitinib in Drug-sensitive and Drug-
resistant Cancer Cell Lines after Arming with a Singlet Oxygen
Releasing Moiety
Kunshan Huang, Beibei Huo, Dongyao Li, Jinping Xue, and Juanjuan Chen*.
K. S. Huang, B. B. Huo, D. Y. Li, Prof. J. P. Xue, Prof. J. J. Chen
National and Local Joint Biomedical Engineering Research Center on Photodynamic Technologies, College of Chemistry
Fuzhou University
Fuzhou 350116, Fujian, China
E-mail: chenjuanjuan@fzu.edu.cn
Abstract: Attractive results have been achieved with small
molecular target-based anticancer drugs in the anticancer field;
however, enhancing their treatment effect and solving the drug-
resistant problem remain key concerns worldwide. Inspired by the
specific affinity of gefitinib for tumour cells and the strong oxidation
capacity of singlet oxygen, we combined a chemically generated
singlet oxygen moiety with the small molecular-targeted drug
gefitinib to improve its anticancer effect. We designed and
synthesised a novel compound (Y5-1), in which a small molecular
targeted therapy agent (gefitinib) and a singlet oxygen (provided by
an in vitro photodynamic reaction) thermal-controlled releasing
moiety are covalently conjugated. We demonstrated that the
introduction of the singlet oxygen thermal-controlled releasing
moiety enhanced the anticancer activities of gefitinib. The results of
this study are expected to provide a novel strategy to enhance the
effect of chemotherapy drugs on drug-resistant cell lines.
Much effort has been devoted to developing novel small
molecular-targeted anticancer drugs or other new strategies to
improve the anticancer treatment effect of gefitinib and
overcome resistance.[8] One such strategy is to design new-
generation TKIs such as afatinib and avitinib;[9] unfortunately,
most of them have been found to have poor clinical patient
outcomes or serious side effects such as hyperglycaemia.[10]
Another approach is the combination of gefitinib with other
drugs such as docetaxel, bisphosphonate zoledronic acid, and
chloroquine diphosphate;[11] however, this approach is difficult
to apply in clinical practice and may result in more side effects.
Another method is to use a nanocarrier for gefitinib and other
anticancer drugs; for example, functionalised graphene oxide
has been used as a nanocarrier for gefitinib and quercetin.[12]
However, the dosage of anticancer drugs is not accurate.
Photosensitisers are considered controllable and safe
anticancer drugs.[13] During photodynamic therapy, singlet
oxygen, which is produced by energy transfer from an excited
photosensitiser to oxygen, can induce oxidative damage to
Introduction
cellular components (e.g. proteins, lipids, and DNA), apoptosis,
or necrosis.[14] For several years, our research group has
designed and synthesised many novel anticancer drugs.[15] We
Cancer is the second leading cause of death and has become a
huge global health burden.[1] The discovery and development of
novel and efficient anticancer drugs or strategies for cancer
treatment are the most important goals in modern medicinal
chemistry.[2] In clinical trials, small-molecule inhibitors of
tyrosine kinase (TKI) represent a major advance in cancer
treatment.[3] The first rationally designed small-molecule
inhibitor, imatinib (Gleevec®, Novartis), was approved by the
FDA for the therapy of chronic myeloid leukaemia (CML) in
have conjugated small molecular-targeted drugs such as
gefitinib with a phthalocyanine core and demonstrated that
these conjugates are highly promising antitumor agents.[15]
However, these agents face several challenges, including
therapy dosimetry and reliance on oxygen, light, and PSs.[16]
Based on the specific affinity of gefitinib for tumour cells and the
strong oxidation capacity of singlet oxygen, we hypothesised
that combining a chemical source of singlet oxygen with
gefitinib in a single molecule would improve the anticancer
effect of gefitinib. Introducing chemically generated singlet
oxygen into small molecular-targeted drugs would be of great
interest and possible clinical value.
2
001, and this development is considered to have started a
new era in anticancer drug discovery.[4] Subsequently, more
than 30 small molecular-targeted drugs have been approved for
clinical application to treat different types of human cancers.[5]
Gefitinib, a small molecular-targeted anticancer drug, can target
the ATP-binding domain of tyrosine kinase in epidermal growth
factor receptor (EGFR)-overexpressing tumours and has
exhibited specific affinity for tumour cells.[6] However,
accumulating clinical evidence suggests an unsatisfactory
treatment effect and drug resistance.[7]
A perfect source of 1O2 should have outstanding singlet
oxygen yields and release singlet oxygen under mild conditions.
The endoperoxides of 2-pyridone and its derivatives have been
shown to have outstanding singlet oxygen yields and release
singlet oxygen under body temperature.[17] They can induce the
apoptosis of cancer cells by the thermal release of 1 .[17,18]
O
2
1
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Moreover, endoperoxides can be formed by the reaction
accurate therapy dosimetry, high selectivity, and anticancer
1
and 2-pyridone.[17,18]
between O
2
activities, Y5-1 may be applied in clinical practice.
Inspired by the considerable efforts to develop more effective
and safer anticancer drugs, we designed and synthesised a
novel compound (Y5-1), in which 2-pyridone endoperoxides as
Results and Discussion
a 1
2
O provider was conjugated with gefitinib units by a click
The synthesis of compounds Y5-1 and Y5 is shown in Scheme
reaction. Y5-1 displays high affinity for and anticancer activities
against cancer cells overexpressing EGFR. The results of this
study may lead to improvements in the therapeutic efficacy of
not only small molecular-targeted anticancer drugs but also
chemotherapeutic drugs in MDR cancer cells. Based on its
1
. First, Y1, Y2, and Y2-1 were synthesised by a previously
published method.[19] Y4 was synthesised by a nucleophilic
reaction between gefitinib and bromopropyne. Subsequently,
Y5-1 was synthesised by the click reaction of Y2-1 and Y4 with
CuSO
4 2
·5H O and L-ascorbic acid sodium salt as catalysts. Y5
was synthesised in a simple and similar manner.
Scheme 1. Synthesis of Y5-1 and Y5.
An 1O
used to test the 1
scavenger (1,3-diphenylisobenzofuran, DPBF) was
-releasing capacity of Y5-1. As shown in
release O
showed a slight downtrend of DPBF at 25 °C (Figure 1b),
demonstrating that the 1
-releasing capacity of Y5-1 greatly
1
2
2
at 37 °C (Figure 1a). In addition, the Y5-1 contrast
O
2
Figure 1, the absorbance intensity of DPBF (Y5-1 and the
DPBF group) decreased at body temperature (37 °C), while the
downtrend in the blank contrast and Y5 contrast was negligible,
demonstrating that the endoperoxide structure of Y5-1 can
O
2
decreases at 25 °C. These results indicate that Y5-1 can slowly
1
release O
2
at body temperature (37 °C).
2
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10.1002/cmdc.202000036
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Figure 1. a) Spectrum changes of DPBF at 415 nm during the determination of singlet oxygen for Y5 and Y5-1 at 37 °C; b) Spectrum changes of DPBF and Y5-1
at 415 nm vs time (25 °C and 37 °C); inset: DPBF, Y5 (1.5 mmol), and Y5-1 (1.5 mmol) were all dissolved in DMF.
The anticancer effects of Y5-1, Y5, and gefitinib were evaluated
using the methyl thiazolyl tetrazolium (MTT) assay, in which
three cancer cell lines, A549 cells (high EGFR expression),
HepG2 cells (high differentiation and proliferation), and H1975
cells (gefitinib-resistant) were used. The dose-dependent
survival curves are shown in Figure 2, and the corresponding
IC50 values are listed in Table 1. The significant differences in
the anticancer effects of Y5 (with an IC50 = 51.3-94.2 μM) and
Y5-1 (IC50 = 9.2-14.8 μM) on these three cancer cell lines,
demonstrating that conjugating gefitinib with endoperoxide
clearly enhanced its anticancer effects. As expected, Y5-1, Y5
armed with 1O
gefitinib. These results clearly showed that introduction of an
1O
-releasing moiety into the gefitinib structure significantly
2
, had 2.6-4.2-fold higher cytotoxicity than
2
enhanced its anticancer effects, and Y5-1 had the highest
cytotoxicity in gefitinib-resistant cancer cells.
Figure 2. Cell toxicity of gefitinib (orange), Y5 (grey), and Y5-1 (red) on A549, HepG2, and H1975 cells (data are expressed as the mean value ± standard error of
the mean value of four independent experiments).
Table 1. Cellular antiproliferative activities towards different cancer cells.
The antiproliferative activity of Y5-1 towards cancer cells is
associated with not only cellular uptake but also singlet oxygen
Cellular Antiproliferative Activity (IC50, μM)
production. To further explain the differences in the anticancer
effects of Y5-1 against these three cell lines, cellular uptake
compounds
and singlet oxygen generation efficiency were evaluated in
A549
HepG2
H1975
detail. First, absorption and fluorescence spectroscopy were
used to study the cellular uptake of Y5-1. The average uptake
data are shown in Figure 3a. The uptake of Y5-1 in A549 cells
was significantly higher than that in HELF cells, HepG2 cells,
and H1975 cells. These results indicate that the introduction of
the gefitinib moiety into Y5-1 imparted affinity for cancer cells
with high EGFR expression and higher sensitivity to gefitinib.
Second, DCFH-DA (2',7’’-dichlorodihydrofluorescein diacetate)
was used to measure ROS generation because the singlet
Gefitinib
Y5
38.0
51.3
14.8
28.0
94.2
9.2
45.6
60.1
10.8
Y5-1
3
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oxygen produced by Y5-1 is a type of ROS. Moreover, the ROS
level is an important parameter that reflects the anti-tumour
effect. Therefore, we selected DCFH-DA to detect the
production of ROS by Y5-1. As shown in Figure S1 (Supporting
Information), for both HepG2 cells and A549 cells, the ROS
generation in the Y5-1 group was positively correlated with the
concentration of Y5-1, and there was no significant difference in
the ROS production efficiency of Y5-1 between these two cell
lines at the same concentrations. Compared with Y5 and
gefitinib, Y5-1 can release significantly more ROS in HeLa cells
(Figure 3b). Therefore, the higher cellular uptake and ROS
generation of Y5-1 make it an excellent anticancer drug.
Figure 3. a) Uptake of Y5-1 in different cell lines. b) Generation of ROS in HeLa cells with no addition, gefitinib (25 μM), Y5 (25 μM), and Y5-1 (25 μM) (there were
six independent experiments for each group; statistical analysis: ***p < 0.001, **p < 0.01, *p < 0.05, ns indicates p > 0.05).
4
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and CuSO
solvent (tBu-OH/H
at °C. The product was purified by column chromatography
CH OH/CH Cl from 1:100 to 1:40) to acquire compound Y5-1 (0.0478
g, 7.96%) as a white solid. 1H NMR (500 MHz, chloroform-d) δ 8.60 (s,
H), 7.50 (s, 1H), 7.34 (dd, J = 6.5, 2.4 Hz, 1H), 7.28 (s, 2H), 7.24 (s,
4
·5H
2
O (0.0252 g, 0.1010 mmol) were dissolved in a mixture
Conclusion
2
O/THF, 4:2:8 mL). The solution was stirred for 24 h
3
We designed and synthesised a novel compound (Y5-1) and
evaluated its in vitro anticancer activities. This compound
contains a small molecular-targeted agent (gefitinib) and a
singlet oxygen (added in an in vitro photodynamic reaction)
thermal-controlled releasing moiety, which are covalently linked
and act in a cooperative manner. We demonstrated that Y5-1
(
3
2
2
1
1H), 7.21–7.16 (m, 3H), 7.12 (s, 2H), 7.12 (s, 2H), 6.30 (s, 1H), 5.77 (d,
J = 6.1 Hz, 1H), 5.56–5.52 (m, 1H), 5.47 (q, J = 4.8, 4.0 Hz, 1H), 5.41 (d,
J= 9.2 Hz, 1H), 5.40–5.34 (m, 3H), 5.16 (s, 1H), 5.13 (s, 1H), 5.05 (s,
1
H), 5.01 (d, J =9.3 Hz, 1H), 4.23 (dd, J = 15.1, 7.3 Hz, 1H), 4.18 – 4.14
(m, 1H), 3.96 (s, 1H), 3.93 (d, J= 3.1 Hz, 3H), 3.71 (d, J = 4.1 Hz, 1H),
.25– 2.21 (m, 1H), 2.19 (s, 1H), 2.03 (q, J = 6.6 Hz, 1H), 1.83 (s, 3H).
The compound decomposes; thus, its 13C NMR data are not available.
HRMS (ESI): m/z calcd 42ClFN [M+CH
COOH]+: 817.2871,
found: 817.2870, Δ=0.13 ppm.
exhibits high specific affinity for EGFR-overexpressing cancer
1
cells. Additionally, the introduction of the O
2
thermal-controlled
2
releasing moiety clearly enhanced the anticancer activities of
gefitinib in drug-resistant cell lines. To enable the use of Y5-1
for clinical applications, more detailed studies on its anti-tumour
mechanisms and in vivo animal experiments are in progress.
Overall, this work presents not only a novel compound with
efficient anticancer activity but also a design strategy for
developing high-efficiency anticancer drugs.
C
40
H
8
O
8
3
1
) Release Experiments. The release of 1
2
O was
Singlet Oxygen ( O
2
1
tested using 1,3-diphenylisobenzofuran as a
solutions of Y5 and DPBF or Y5-1 and DPBF were placed in water at
5 °C or 37 °C and kept in the dark. The absorption spectra were
O2 quencher. The mixture
2
measured at 415 nm.
Experimental Section
Cytotoxicity Experiment. First, 5000 A549, H1975, and HepG2 cells
were seeded into each well of a 96-well plate and incubated 24 h. The
deliquescent drugs (gefitinib, Y5, and Y5-1) were diluted with culture
medium to different concentrations, and the drugs were then added to
the plate. Every concentration had six replicates. After incubation for 24
All reagents were purchased from commercial sources. 13C NMR and H
1
NMR spectra were recorded on a nuclear magnetic resonance device
with AVANCE III (400 MHz) or AVANCE III 500 (500 MHz). DMSO-d
6
and CDCl were used as deuterated reagents. HRMS data were
3
h, pre-prepared MTT (10 μL, 5 μg/μL in H
2
O) was added to the 96-well
for 4 h. The
obtained using LC-QTOF-MS (G6520B). The product purity was greater
than 95%. A Hitachi 4510-UV-Visible Absorption Spectrometer was
plate followed by incubation at 37 °C under 5% CO
2
formazan was dissolved in dimethylsulfoxide (100 μL/well), and the pate
was shaken for 30 min. Absorbance at 570 nm was measured using a
SpectraMax@ i5x.
used to detect the 1
2
O released by Y5-1. The anticancer activity of Y5-1
was evaluated from three aspects, including cytotoxicity, cellular uptake,
and ROS production ability. The remaining instruments included a
spectraMax@ i5x.
Cellular Uptake. First, 5000 HELF, A549, H1975, and HepG2 cells
were seeded into a 96-well plate and incubated at 37 °C for 24 h. Each
experiment was repeated six times. Then, 100 μL medium containing a
drug at 25 μM was added to each well and incubated for 24 h. The plate
was washed with physiological saline solution three times to remove the
drugs. Then, the cells were lysed with 1% SDS solution (120 μL). The
uptake of drugs was detected with a plate reader (excitation at 335 nm,
emission at 481 nm). The quantity of cellular protein was measured
using a BCA Protein Assay Kit.
Synthesis of Compound Y4. Gefitinib and propargyl bromide were
dissolved in N,N-dimethylformamide (10 mL) and stirred at 37 °C for 25
h. The product was purified by column chromatography (CH
3 2 2
OH/CH Cl
from 1:100 to 1:40). Compound Y4 was obtained as a yellow solid
(
(
0.3087 g, 26.08%). 1H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 7.64
dd, J = 6.3, 2.3 Hz, 1H), 7.47 (t, J =9.0 Hz, 1H), 7.22 (d, J = 3.8 Hz, 2H),
6
6
2
1
1
5
.38 (s, 1H), 4.85 (s, 2H), 3.90 (s, 3H), 3.60 – 3.55(m, 4H), 3.51 (t, J =
.5 Hz, 2H), 3.15 (s, 1H), 2.30 (d, J = 7.9 Hz, 4H), 1.69 (p, J = 6.4 Hz,
H), 1.31–1.21 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 159.14,
54.63, 152.94, 149.32, 147.41, 143.39, 128.56, 127.51, 127.45, 118.29,
18.12, 110.51, 107.82, 105.16, 80.35, 75.41, 66.61, 66.56, 56.35,
Cellular ROS Production Assessment. ROS in cells was detected
using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as a probe
because it can react with ROS and generate the fluorescent substance
DCF. The cancer cells were seeded into a black 96-well plate at 5000
cells per 100 μL and incubated for 24 h. The old culture medium was
removed, each well was washed with physiological saline solution, and
new medium with a drug at a concentration of 25 μM was added. A
control group of feminine and masculine was established. After
incubation for 24 h, the black 96-well plate was washed with
physiological saline solution and incubated with 40 μL of DCFH-DA (10
μM). After incubation for 30 min, the plate was washed with
physiological saline solution four times, and the cells were lysed with
5.37, 55.01, 53.80, 42.55, 25.62. HRMS (ESI) m/z calcd
+
C
25
H
26ClFN
4
O
3
[M+H] : 484.1677, found : 485.1756.
Synthesis of Compound Y5. Compound Y2 (0.1655 g, 0.689 mmol),
Y4 (0.3146 g, 0.649 mmol), sodium ascorbate (0.0202 g, 0.1018 mmol),
and CuSO
reagent (tBu-OH/H
at 37 °C. The product was purified by column chromatography
CH OH/CH Cl from 1:100 to 1:20) to acquire Y5 (0.2582 g, 57.57%)
as a light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H),
.16 (s, 1H), 7.73 (d, J = 6.5 Hz, 1H),7.55 (d, J = 6.4 Hz, 1H), 7.39 (q, J
9.1, 7.9 Hz, 2H), 7.25 - 7.17 (m, 4H), 7.14 (d, J =7.8 Hz, 2H), 6.43–
4
·5H
2
O (0.0253 g, 0.1012 mmol) were dissolved in a mixture
2
O/THF, 4:2:8 mL). The mixture was stirred for 23 h
(
3
2
2
120 μL of 1% SDS for 30 min. The fluorescence of DCF was measured
8
using a plate reader (excitation at 488 nm, emission at 525 nm).
=
6
2
1
1
1
1
5
7
.35 (m, 2H), 6.21 (t, J = 6.7 Hz, 1H), 5.49 (s, 2H), 5.33 (s, 2H), 5.05(s,
H), 3.90 (s, 3H), 3.58 (s, 4H), 3.47 (t, J = 6.4 Hz, 2H), 2.32 (s, 4H),
.73–1.63 (m, 2H), 1.23 (s, 2H).13C NMR (101 MHz, DMSO-d6) δ
61.81, 159.30, 154.51, 153.00, 147.20, 143.75,140.51, 139.50, 137.68,
35.93, 128.60, 128.41, 128.22, 127.44, 127.37, 125.00, 120.33, 118.21,
18.00, 107.87, 105.97, 105.47, 66.56, 66.54, 56.34, 54.99, 53.76,52.81,
Acknowledgements
We thank the National Health and Family Planning Commission
jointly established scientific research fund (Project No.
WKJ2016-2-14) and the National Natural Science Foundation
of China (Project No. 81703345).
38 8 4
C H38ClFN O
[M+H]+:
1.20, 48.53, 25.62. HRMS (ESI): calcd
25.2761, found: 725.2779, Δ=2.48 ppm.
Synthesis of Compound Y5-1. Compound Y4 (0.3939 g, 0.8081 mmol),
Y2-1 (0.2 g, 0.7346 mmol), sodium ascorbate (0.0198 g, 0.1002 mmol),
5
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Conflict of interest: The authors declare no conflict of interest,
financial or otherwise, in this paper.
Keywords: Drug-resistant • Chemotherapy • Drug design •
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ChemMedChem
10.1002/cmdc.202000036
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Entry for the Table of Contents
we introduce the chemically generated singlet oxygen moiety into gefitinib, gefitinib can inhibit the activity of tyrosine kinase and the
1O
thermal-controlled releasing moiety can obviously enhance the anticancer activities of gefitinib on the drug-resistant cell lines by
2
1
Increasing intracellular O
2
level
.
Institute and/or researcher Twitter usernames: Kunshan Huang, Beibei Huo, Dongyao Li, Jinping Xue, and Juanjuan Chen*
Page No. –Page No.
Enhanced Efficacy of Gefitinib in Drug-sensitive and Drug-resistant Cancer Cell Lines after Arming with a Singlet Oxygen
Releasing Moiety
7
This article is protected by copyright. All rights reserved.