Ferrocenyl-Triphenyltin Complexes as Lysosome-Targeted Imaging and Anticancer Agents

Article abstract of DOI:10.1021/acs.inorgchem.8b03305

In this paper, two ferrocenyl-triphenyltin complexes were synthesized and characterized. Complex 2 is constructed as new multifunctional therapeutic platform for lysosome-targeted imaging and displayed much higher cytotoxicity than its analogue 1 by the introduction of a methyl group instead of a hydrogen atom in acylhydrazone. The cyclic voltammograms and reaction with GSH (glutathione) further confirmed that complex 1 has a reversible redox peak and can react with GSH, which indicate that complex 1 might lose its anticancer effect by undergoing reaction with GSH once it enters the cancer cell. Complex 2 could effectively catalyze the oxidation of NADH (the reduced form of nicotinamide adenine dinucleotide) to NAD⁺ and induce the production of reactive oxygen species (ROS), lead to caspase-dependent apoptosis through damaged mitochondria, simultaneously, accounting for the mitochondrial vacuolization and karyorrhexis. The caspase-3 activation and cytoplasmic vacuolation karyorrhexis induced by complex 2 revealed that the A549 cell lines might undergo cell death primarily mediated by apoptosis and oncosis; however, 1 cannot reproduce this effect. Taken together, these results indicated that complex 2 has more potential for evolution as a new bioimaging and anticancer agent.

Full text of DOI:10.1021/acs.inorgchem.8b03305

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Ferrocenyl−Triphenyltin Complexes as Lysosome-Targeted Imaging
and Anticancer Agents
Juanjuan Li,† Xicheng Liu,*,† Haifeng Zhang,‡ Xingxing Ge,† Yanhua Tang,† Zhishan Xu,†,§
Laijin Tian,† Xiangai Yuan,† Xudong Mao,† and Zhe Liu*,†
†Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis and Key
Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, School of Chemistry and Chemical Engineering,
Qufu Normal University, Qufu 273165, China
‡Department of Chemistry and Chemical Engineering, Jining University, Qufu 273155, China
§Department of Chemistry and Chemical Engineering, Shandong Normal University, Jinan 250014, China
S
* Supporting Information
ABSTRACT: In this paper, two ferrocenyl−triphenyltin
complexes were synthesized and characterized. Complex 2 is
constructed as new multifunctional therapeutic platform for
lysosome-targeted imaging and displayed much higher
cytotoxicity than its analogue 1 by the introduction of a
methyl group instead of a hydrogen atom in acylhydrazone.
The cyclic voltammograms and reaction with GSH (gluta-
thione) further confirmed that complex 1 has a reversible
redox peak and can react with GSH, which indicate that
complex 1 might lose its anticancer effect by undergoing
reaction with GSH once it enters the cancer cell. Complex 2
could effectively catalyze the oxidation of NADH (the
reduced form of nicotinamide adenine dinucleotide) to
NAD+ and induce the production of reactive oxygen species (ROS), lead to caspase-dependent apoptosis through damaged
mitochondria, simultaneously, accounting for the mitochondrial vacuolization and karyorrhexis. The caspase-3 activation and
cytoplasmic vacuolation karyorrhexis induced by complex 2 revealed that the A549 cell lines might undergo cell death primarily
mediated by apoptosis and oncosis; however, 1 cannot reproduce this effect. Taken together, these results indicated that
complex 2 has more potential for evolution as a new bioimaging and anticancer agent.
factors, the toxic side effects of organotin complexes could be
decreased. Previous studies have shown that the toxic side
effects could be decreased by increasing the numbers of alkyl
substituents on the tin atom to a certain extent or using an
alkyl with a long carbon chain to replace the smaller one, such
as butyl, phenyl, benzyl, cyclohexyl, and so on.11
Recent studies showed that classic molecule assembly of
NADH (reduced form of nicotinamide adenine dinucleotide)
and NAD+ lies in various biological processes, including
calcium homeostasis, immunological functions, energy metab-
olism, antioxidation/generation of oxidative stress, mitochon-
drial functions, and so on,12−16 which have been extensively
studied in the field of cell death.17−19 NAD+ and NADH could
adjust various significant factors in cell death, for example,
poly(ADP-ribose) polymerase-1 (PARP-1), mitochondrial
permeability transition, and apoptosis-inducing factor. Simulta-
neously, NADH-dependent reactive oxygen species (ROS)
generation from mitochondria is one of critical oxidation
INTRODUCTION
■
Medicinal application of ferrocene derivatives has been an
active research area in the last few decades. Many reports have
shown that ferrocene derivatives are highly active against
several diseases whether in vitro or in vivo, including fungal
and bacterial infections,1,2 malaria,3 human immunodeficiency
virus (HIV),4 and cancer.5 The anticancer potency of simple
ferrocene derivatives was generally low; however, the
incorporation of the ferrocene group into other transition
metal anticancer complexes, such as platinum, ruthenium,
iridium, copper, and gold complexes, among others, could
effectively enhance their anticancer activity.6 Little efforts have
been put forward to design the combination of ferrocene with
organotin.
Organotin complexes play important roles in biological
activities and human health, and a large number of organotin
complexes have shown anticancer activity higher than that of
the widely used anticancer drug cisplatin in vitro.7−9 It is well-
known that the biochemical activity of organotin complexes is
influenced greatly by the type of groups in the organotin
moiety and the number of Sn−C bonds.10 By tackling these
Received: November 27, 2018
© XXXX American Chemical Society
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mechanisms of anticancer. In addition, numerous studies have
shown that transition metal anticancer complexes, such as
iridium and ruthenium, can catalyze the change of NADH to
NAD+, lead to the production of ROS, and eventually induce
apoptosis.20,21
and red block-shaped single crystals of 1 are also obtained
(CCDC 1843945). The asymmetric unit molecular structure
of complex 1 is shown in Figure 1. Crystal data are listed in
Table S1, and selected bond lengths and angles are listed in
Tables S2, consistent with the expected structure.
Molecular imaging of drugs in cells plays a crucial role in
comprehending their mechanism of action in many significant
cellular processes, such as real-time tracking of drug intra-
cellular transport and monitoring the interactions between the
drug and the biological target molecule. However, the
luminescent properties of organotin complexes are rarely
studied. Also, the development of anticancer complexes for
organelle-specific attack has attracted much attention. Deliver-
ing drugs to organelle-specific sites can effectively increase
drug concentration at the target sites and reduce side
effects.22,23 Now, targeting of the lysosomes has appeared as
an effective means for anticancer therapy due to the ability of
degradation proteins and other cellular organelles.24 Groups
such as those of Mokhir, Chao, and O’Shea have developed
metal complexes and organic compounds as lysosome-tracking
reagent and therapeutic agent.25−27
Figure 1. Asymmetric unit molecular structure with atom numbering
schemes for 1.
The stability of complexes under aqueous and physiological
conditions is one of the most crucial factors in assessing their
applications in the body. Therefore, the stability of as-
synthesized complexes (1 and 2) in 40% MeOH/60% H2O
(v/v) was monitored by ultraviolet−visible (UV−vis) spec-
trum at 298 K (Figure S1). There is no change for the
complexes within 8 h, which indicates that target complexes
have excellent stability under the test conditions. A high level
of reduction for glutathione (GSH, 1 × 10−3−10 × 10−3 M) is
one of the biochemical characteristics of malignant tumor.29
On the basis of these physiological characteristics, the
stabilities of complexes 1 and 2 in 20% MeOH/80% PBS
(v/v, phosphate buffer solution) containing 1 mM GSH was
studied. Interestingly, as shown in Figure S2, complex 1
exhibited an increased absorption after incubation with 1 ×
10−3 M GSH compared with the result in water. However,
complex 2 remained stable under the test conditions within 18
h, which manifests the high stability of complex 2 in
physiological systems outside the tumor sites. Therefore, the
instability with GSH may lead to the loss of anticancer activity
for complex 1.
Inspired by these results, herein, a ferrocene molecule was
introduced into the alkyl chain of triphenyltin through the
acylhydrazone group to increase the length of carbon chain
and the volume of tin complexes. Two iron−tin heteronuclear
metal anticancer complexes were synthesized, and the designed
strategy of target complexes was shown in Scheme 1. It is
Scheme 1. Design Strategy of Ferrocenyl−Triphenyltin
Complexes
Next, the redox properties of 1 and 2 were investigated by
the cyclic voltammetry that performed with two consecutive
scans from −1.0 to +1.0 V. Very different electrochemical
characteristics were observed for complexes 1 and 2. For 1,
reversible redox processes were found to give a very strong
cathodic peak at +0.222 V and an anodic peak at +0.128 V;
however, no obvious redox processes appeared for complex 2
(Figure 2). The results of cyclic voltammograms lead us to
further speculate that complex 1 has favorable redox property,
interesting that the introduction of a methyl group in
acylhydrazone in complex 2 significantly enhanced anticancer
activity. In addition, we investigated the mechanism of action
and evaluated the imaging capabilities of iron−tin hetero-
nuclear complexes.
RESULTS AND DISCUSSION
■
Starting β-methoxycarbonyl ethyl triphenyltin S1 (Scheme 1)
was prepared according to the previously published proce-
dure.28 Subsequently, synthesis of S2 by was achieved with S1
and commercially available hydrazine hydrate. Complexes 1
and 2 were obtained by the reaction of β-hydrazinocarbonyl
ethyl triphenyltin S2 and ferrocene derivatives, and heating at
reflux for 24 h under nitrogen atmosphere. The crude products
were purified by slow diffusion of hexane into saturated
dichloromethane solutions of 1 and 2 at room temperature,
Figure 2. Evaluation of the cyclic voltammograms of 1 and 2 (1 mM)
in anhydrous DMSO solutions, scan rate = 100 mV/s.
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Table 1. Inhibition of Growth of Cancer Cells by 1, 2, and Cisplatin Recorded over a Period of 24 h and log P for Complexes 1
and 2
IC50 (μM)
a
complex
A549
>100
1.4 0.3
HeLa
HepG2
GL261
log P
1
2
67.0 6.3
1.4 0.7
7.5 0.2
69.4 9.6
3.2 0.4
20.9 1.2
30.0 7.9
2.5 0.7
15.4 0.5
2.5 0.5
3.4 0.3
cisplatin
21.3 1.7
a
Results are the means of three independent experiments.
which might lose anticancer efficacy by reaction with GSH
once it enters the cancer cell.
We evaluated the effects of cytotoxicity for 1 and 2 in vitro
against the viability of A549 (human lung cancer cells), HeLa
(human cervical cell cancer), HepG2 (human hepatoma cell),
and GL261 (mouse glioma cells) by MTT assay after 24 h.
The half-maximal inhibitory concentration (IC50) values are
listed in Table 1. Complex 2 is significantly more active than
either cisplatin or 1 in all the cells tested, and 1 has only
slightly antiproliferative effects on GL261 cells. Complex 2
displays cytotoxicity against A549 cells, with an IC50 value of
1.4 0.7 μM, up to a 15-fold increase compared with that of
cisplatin (IC50: 21.3 1.7 μM). Interestingly, the anticancer
activity of complex 2 was significantly increased in all the cells
tested by simply replacing a hydrogen atom by a methyl group
in the acylhydrazone group of 1. L1 and L2 (Scheme S1) are
the analogues of complexes 1 and 2 but without the
triphenyltin unit; S2 is an analogue of as-synthesized
complexes but without the ferrocene unit. As shown in
Table S3, the lower antiproliferative activities of L1, L2, and S2
indicate the integrations of triphenyltin, ferrocene derivative,
and acylhydrazone cause favorable antiproliferative activity for
complex 2. Lipophilicity is one of the significantly factors
which would affect the cytotoxic potency and cellular uptake
levels of the drugs. The lipophilicity of complexes could be
evaluated by the value of log P (partition coefficient in oil/
water). The log P values of 1 and 2 were 2.5 0.5 and 3.4
0.3 respectively (Table 1), which show that the higher the
lipophilicity for complex 2, the better the cytotoxic activity.
Next, the antiproliferative activities of 1 and 2 against BEAS-2B
(human bronchial epithelial normal cell) were evaluated by
MTT assay after 72 h incubation; unfortunately, no obvious
selectivity was observed for complexes 1 and 2.
Figure 3. UV−vis spectra of the reaction of NADH (100 μM) in 50%
MeOH/50% H2O (v/v) at 298 K for 9 h. (a) 1 (1 μM); (b) 2 (1
μM).
S5). The positive charge of the carbon makes the hydride
transfer from the coenzyme NADH reaction more active.
Subcellular localization of 1 and 2 was easily determined by
confocal microscopy in A549 cells on account of their intrinsic
luminescence, which can provide more hints for the
mechanisms of anticancer (Figure 4). The results show that
Biological processes including energy metabolism, cell death,
and mitochondrial function are regulated by NAD+ or NADH
in some manners.30 It has been demonstrated that IrIII and RuII
cyclopentadienyl complexes can be used as highly effective
catalyst to convert the coenzyme NAD+/NADH couple
through transfer hydrogenation reactions. We used UV−vis
and NMR to define the contributions of 1 and 2 to catalyze
NADH to NAD+, a shown in Figures 3 and S3. NADH was
incubated in the same solution (50% MeOH/50% H2O (v/v)
as control (Figure S4). The conversion of NADH to NAD+
was evaluated based on the fact that NADH has a UV
absorption at 339 nm while NAD+ does not. Subtracting the
effects of control, the turnover numbers (TONs) of 2 (84.0)
were calculated at 340 nm. Almost no conversion was detected
for 1, suggesting complex 2 had a strong catalytic ability of to
convert the coenzyme NAD+/NADH couple, but that complex
1 did not. Due to the push-electron effect of the methyl group,
the electron cloud density of CN increased; therefore, the
activity is strengthened. The charge of C (CN) is +0.246 for
complex 2, about 5 times higher than that of 1 (+0.049, Figure
Figure 4. (a) Confocal microscopy images of A549 cells co-labeled
with I (10 μM, 0.5 h) and 2 (IC50 = 1.4 μM, 0.5 h) and LTDR (100
nM, 0.5 h); (b) Confocal microscopy images of A549 cells co-labeled
with I (10 μM, 0.5 h) and 2 (1.4 μM, 0.5 h) and MTDR (75 nM, 0.5
h). The excitation and emission bands were as follows: for complexes:
λex= 488 nm, λem= 520 30 nm; for MTDR: λex = 644 nm, λem= 700
30 nm; for LTDR: λex= 594 nm, λem= 630 30 nm. Scale bar: 20
μm.
C
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1 and 2 specifically targeted lysosomes with high Pearson’s
colocalization coefficients (PCC) of 0.90 and 0.92, respec-
tively, indicating the complexes both are able to stain
lysosomes specifically and possess lysosome-targeting specific-
ity in A549 cells. However, minimal overlap with MTDR
(mitochondria specific probe) is observed for 1 (PCC = 0.04)
and 2 (PCC = 0.06) (Figure 4b). Selective accumulation of the
1 and 2 in cancer cells is of great concern for targeted cancer
therapy.31
Excessive generation of ROS and lysosomal permeability is
often triggered by the disruption of the lysosomal integrity.32,33
To investigate whether lysosomal damage induced by 1 and 2
was accompanied by lysosome-targeting specificity, lysosomal
integrity of A549 cells was evaluated after treatment with 1 (10
μM) and 2 (1.4 μM, IC50) at 1, 6, and 17 h by acridine orange
(AO) staining. AO exhibits red fluorescence when accumu-
lated in lysosomes and green fluorescence when bound to
RNAs in the nuclei and cytosol. As shown in Figure 5, red
fluorescence basically disappears from the lysosome when
exposed to complexes 1 (10 μM) and 2 (1.4 μM) after 1 h
versus control, and obvious lysosomal damage was found after
17 h.
To assess the mechanism of cell death in A549 cells induced
by 1 and 2, we evaluated the cytotoxicity in the presence of
different chemical inhibitors to adjust canonical form of cell
death. The cells were treated with necroptosis inhibitor
necrostatin-1 (Nec-1), autophagy inhibitor 3-methyladenine
(3-MA), the protein synthesis inhibitor cycloheximide (CHX),
and the protease inhibitor leupeptin (LPT) (Table S4). The
data analysis indicates the cytotoxic effects of 1 and 2 did not
demonstrate significantly change, suggesting that these
inhibitors are not operative.
Apoptosis is one of the canonical forms of cell death.34 In
order to further insight into whether 1 and 2 inhibited cancer
cells growth via inducing apoptosis, A549 cells were exposed to
1 and 2 at different concentrations including 0.7, 1.4, 2.8, and
4.2 μM for 24 h and analyzed by flow cytometry. As shown in
Figure 6 and Table S5, almost no apoptosis was induced by 1
even at very high concentrations of 4.2 μM, and 91.8% of cells
remained viable. However, for complex 2, about 68.6% of the
A549 cells were undergoing apoptosis after 24 h at the
concentration of 4.2 μM (including 42.9% of cells in late
apoptosis), while the 92.8% of untreated cells remained viable
under the same conditions.
Studies have confirmed that NADH depletion is an early
event in apoptosis and could mediate cellular energy
metabolism, which is a significant factor to decide cell death
patterns.17,35 To assess whether complex-induced apoptosis is
caused by depletion of NADH in cells, we added additional
NADH when testing for apoptosis. In the presence of
additional NADH, complex 2 appeared to lower the cell
death of human A549 cancer cells to a certain degree
compared to untreated-NADH cells (Figure S6 and Table
S6). As shown, about 69.4% of the treated-NADH A549 cells
were viable; however, about 31.4% were viable when only
treated by complex 2 at a concentration of 4.2 μM. The higher
Figure 5. Observation of lysosomal disruption in A549 cells caused by
1 (10 μM) and 2 (1.4 μM) and stained by AO (5 μM, 15 min). λex
=
488 nm, λem = 510 20 nm; λem = 625 20 nm. The cells were
treated with (a) control, (b) 1 (10 μM) 1 h, (c) 1 (10 μM) 6 h, (d) 1
(10 μM) 17 h, (e) 2 (1.4 μM) 1 h, (f) 2 (1.4 μM) 6 h, (g) 2 (1.4 μM)
17 h. Scale bar: 20 μm.
Figure 6. Flow cytometry detected apoptosis based on annexin V and PI staining of A549 cells treated with 1 and 2 after 24 h at 310 K. (a)
Populations for cells treated by 1 and 2. (b) Histogram for A549 cells treated with different concentrations of 1 and 2 for 24 h.
D
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cell viability indicated that the presence of additional NADH
obviously protected the cells from the attack of complex 2, thus
showing that depletion of NADH has a critical function in
inducing apoptosis.
karyorrhexis were observed by 2 at 4.2 μM after 0.5 h (Figures
8a and S7). In a time- and dosage-dependent manner a large
The intrinsic pathway of apoptosis is regulated by
mitochondria through the release of proapoptotic proteins
from mitochondria to activate caspases (death-driving
proteolytic proteins).36 Caspase-3 is an important biomarker
of apoptosis which has been identified as a key performer of
apoptosis in mammalian cells.37 The activation of caspase-3 in
A549 cells was verified using the Caspase-Glo assay which was
operated to elucidate the effects of 1 and 2 on caspase-3
activity. As shown in Figure 7 and Table S7, for complex 2, an
Figure 8. Representative confocal microscopy images showing the
morphological features of A549 cells treated with 2 at 4.2 μM for 0.5
and 1 h. (a) 2 4.2 μM, 0.5 h, (b) 2 4.2 μM, 1 h, (c) 2 4.2 μM, 0.5 h
and 3-MA, (d) co-labeled with 2 (4.2 μM, 0.5 h) and MTDR (75 nM,
0.5 h). The excitation and emission bands were as follows: for
complex: λex= 488 nm, λem= 520 30 nm; for Hoechst: λex = 405 nm,
λem= 450 30 nm; for MTDR: λex = 644 nm, λem= 700 30 nm.
Scale bar: 20 μm.
number of karyorrhexis appeared and scattered in the
cytoplasm accompanied by serious cytoplasmic vacuolation.
The autophagy inhibitor 3-methyladenine (3-MA) was used to
prove whether cell vacuolization was caused by autophagy; the
result showed the cytoplasmic vacuolation induced by 2 in
A549 cells was not influenced by 3-MA, hinting a pathway
different from the autophagy proces (Figure 8c). Hollow
spheres of abnormal morphology represent the extremely
swollen and damaged mitochondria in most cells, suggesting
mitochondria dysfunction.40 After 0.5 h of incubation of A549
cells with 2 (4.2 μM), the morphological changes were
observed by damaged mitochondria, Figure 8d. Co-localization
analysis of 2 and MTDR gives proof that most mitochondria
show hollow spheres, representing the extremely swollen
mitochondria. Mitochondria dysfunction mediated forms of
cell death are characterized by these morphological changes.
Increased mitochondrial membrane permeability in ex-
tremely swollen and damaged mitochondria of A549 cells are
regarded as early events.36 The loss of mitochondrial
membrane potential (MMP) induced by 1 and 2 can be
assessed by detecting the decrease in red fluorescence and
increased green fluorescence using JC-1 and flow cytometry. At
the indicated concentrations, 2 displayed a significant
concentration-dependent increase with a remarkable loss of
MMP, e.g., from 8.7% (control) to 32.9% (2.8 μM); however,
1 caused negligible impact on the MMP level (Figure 9a and
Table S9). Studies have shown that the reductive coenzyme of
cytosolic NADH could be directly transferred to oxygen in the
mitochondria with the generation of electrochemical mem-
Figure 7. Detection of caspase-3 activity in A549 cells after the cells
were treated with different concentrations of 1 and 2. (a) FL2
histogram the populations of cells in activated caspase-3 after
treatment of different concentrations of 1 and 2 for 24 h. Data are
quoted as mean
SD of three replicates. (b) Flow cytometry
detected apoptosis based on Annexin V/PI assay of A549 cells treated
with 2 and the pan-caspase inhibitor Z-VAD-FMK for 24 h.
increase in caspase-3 activity of about 29-fold was observed,
compared with that of control, and the activation of caspase-3
was caused in a dose-dependent manner. However, the
activation of caspase-3 induced by 1 is not observed (Figure
7a). Next, the apoptosis caused by 2 was efficiently held back
when adding the caspase inhibitor z-VAD-fmk (5 μM); the cell
viability remains above 80% (Figure 7b and Table S8).
Oncosis is different from apoptosis in morphological
changes, which might hold the key of overcoming drug-
resistance in cancer therapy. Oncosis is characterized by a
series of defined morphological events, such as cytoplasmic
vacuolation, karyorrhexis, or even dissolution, and mitochon-
drial swelling.38,39 As shown in Figure 4, weak cytoplasmic
vacuolation and solid granules have been induced by 2 at 1.4
μM after 0.5 h as shown in confocal microscopy images of
A549 cells; however, this result was not observed with 1. To
gain more insights into the intracellular effects of 2, the
mitochondria and nuclear morphological changes were further
studied using confocal microscopy. Substantial morphological
changes including cytoplasmic vacuolation and partial
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Figure 9. (a) Representative histograms for the analysis of MMP by flow cytometry using the JC-1 dye. The red aggregates and green monomers
are gated. (b) MMP is analyzed by flow cytometry in A549 cancer cells treated with complex 2 at different concentrations and complex 2 in the
presence of NADH (400 μg/mL) at 2.8 μM. Data are quoted as mean SD of three replicates.
brane potential under certain conditions, which are mediated
by cytosolic cytochrome c and mitochondrial cytochrome
oxidase.41,42 Complex 2 may break this process by consuming
NADH in the cell, causing a decrease in MMP. Therefore, we
add NADH to the cell solution containing complex 2 at a
concentration of 2.8 μM, which only causes 1.5% of cells to
lose MMP. As shown in Figures 9b and S8 and Table S10, the
lower loss of MMP in NADH-treated cells indicated that
exogenous NADH effectively prevents the increase of
mitochondrial membrane permeability induced by complex 2.
Meanwhile, NAD may also affect the antioxidation and
generation of oxidative stress through several pathways.12 The
capability of mitochondrial oxidative stress for 1 and 2 was
detected using DCFH-DA fluorescence assay by flow
Figure 10. ROS induction analyzed by flow cytometry in A549 cancer
cells treated with complex 2 at different concentrations and complex 2
in the presence of NADH (400 μg/mL) at 2.8 μM. Data are quoted as
mean SD of three replicates.
cytometry analysis. As shown in Figure S9 and Table S11,
ROS generated in A549 cells exhibit the concentration-
dependent increase for complex 2, but 1 did not observe any
change. At 2.8 μM, complex 2 displayed a very strong ROS-
inducing ability compared with that of negative control,
However, for complex 1, almost no change occurred even at
2.8 μM, which showed that ROS contributes to cell death of
A549 cells. The ROS generation can cause cell death through
As NADPH oxidase is one of the main sources of ROS,46,47
the effect of complex 2 on NOX4 was subsequently tested.
Studies have shown that vast amounts of ROS were generated
through the activation of the membrane-associated ROS-
mitochondria dysfunction and autophagic vacuole accumu-
producing enzyme NADPH oxidase (NOX).48 The results
lation.43 The previous study showed that xanthine oxidase/
display that the expression of NOX4 was significantly
enhanced after complex 2 (2.8 μM) treatment for 24 h,
suggesting that 2-induced ROS generation in A549 cells was
mediated by activating NOX4 (Figure S11).
xanthine dehydrogenase could induce the generation of ROS
by oxidizing NADH.44,45 To probe the oxidizing capacity of
the generation of ROS, A549 cancer cells were pretreated with
NADH (400 μg/mL) before the addition of complex 2
(Figures 10 and S10 and Table S12). Compared with the result
without NADH, a certain degree of decrease in the generation
ROS was observed for cells pretreated with NADH for 2 at the
concentration of 2.8 μM. It maybe due to excessive NADH
consuming the ROS produced by complex 2.
The cytotoxicity inducted by anticancer drugs could be
associated with arresting cell cycle. The effects of 1 and 2 on
cell cycle distribution were investigated by flow cytometry
analysis (Figure S12 and Table S13). The percentages of A549
cells have a small increase in the S phase from 25.5 to 31.9%,
indicating that 1 has a weak disturbance at the S phase of the
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5.7, 2.6 Hz, 6H), 7.38−7.35 (m, 9H), 2.50 (t, J = 7.8 Hz, 2H), 1.67 (t,
J = 7.8 Hz, 2H); ESI-MS (m/z): [M + Na]+ Calcd for C21H22N2OSn:
461.08. Found 461.25.
cell cycle, and that 2 disturbed the G1 phase that increased
8.9%. The results indicate that negligible cycle arrest is not the
cause of the difference in cell death caused by complexes 1 and
2.
Synthesis of Ferrocenyl−Organotin Compounds (1 and 2).
The β-hydrazinocarbonyl ethyl triphenyltin (S2, 2.0 g, 4.3 mmol) and
ferrocene derivatives (4.5 mmol) was dissolved in methanol in a dry
round-bottomed flask and refluxed for 24 h. The progress of reaction
was monitored by TLC. After complete conversion, methanol was
removed under reduced pressure and product was dissolved in
dichloromethane and filtered through Celite filtration funnel and
recrystallized by slow diffusion of n-hexane in a concentrated solution
of the compound in dichloromethane to obtain the corresponding
complexes (1 and 2)
CONCLUSION
■
In summary, we have synthesized and characterized two
ferrocenyl−triphenyltin complexes acting as lysosome-target-
ing multifunctional platforms for anticancer therapy agents.
Subtle structural changes lead to a significant impact on the
cytotoxicity; 2 displayed higher cytotoxicity than 1 and
cisplatin against A549, HeLa, HepG2, and GL261 cells. The
results observed in the cyclic voltammograms and reaction
with GSH for complexes 1 and 2 further confirmed that
complex 1 showed favorable redox property but might lose
anticancer effect by undergoing reaction with GSH. As an
effective catalyst, 2 could convert NADH to NAD+ through
transfer hydrogenation reactions. NADH plays a significantly
role in the loss of MMP and inducing ROS generation.
Complex 2 could consume NADH in the cells, causing a
decrease in mitochondrial membrane potential, have a
significantly influence on ROS, and ultimately leading to cell
death. The results of caspase-3 activation and cytoplasmic
vacuolation karyorrhexis induced by 2 revealed that the A549
cell line might undergo cell death primarily mediated by
apoptosis and oncosis; however, 1 cannot reproduce this effect.
Taken together, various anticancer mechanism tests showed
that complex 2 had good activity, which gives it more potential
for evolution as new bioimaging and anticancer agents.
1: yield: 70.5%. 1H NMR (500 MHz, CDCl3) δ 9.15 (s, 1H), 7.61
(dd, J = 6.3, 2.9 Hz, 5H), 7.36 (dd, J = 4.8, 1.7 Hz, 10H), 4.46−4.44
(m, 2H), 4.34−4.32 (m, 2H), 4.12 (s, 5H), 3.06 (t, J = 7.9 Hz, 2H),
1.73 (t, J = 7.9 Hz, 2H). Anal. Calcd: C, 60.70; H, 4.78; N, 4.42.
Found: C, 60.74; H, 4.72; N, 4.43. ESI-MS (m/z): [M − Ph]+ Calcd
for C32H30FeN2OSn: 634.07. Found: 556.89.
2: yield: 65.3%. 1H NMR (500 MHz, CDCl3) δ 8.71 (s, 1H), 8.49
(s, 1H), 7.84−7.73 (m, 6H), 7.44−7.31 (m, 9H), 4.61−4.57 (m, 2H),
4.41−4.38 (m, 2H), 4.16 (d, J = 5.2 Hz, 5H), 3.24 (dd, J = 13.1, 5.4
Hz, 2H), 2.10 (s, 3H), 1.81 (t, J = 7.7 Hz, 2H). Anal. Calcd: C, 61.24;
H, 4.98; N, 4.33. Found: C, 61.26; H, 4.94; N, 4.43. ESI-MS (m/z):
[M − Ph]+ Calcd for C33H33FeN2OSn: 648.09. Found: 570.90.
L1 and L2 were synthesized by propionic acid hydrazine and
ferrocene derivatives using the same methods of ferrocenyl−organotin
compounds (Scheme S1).
L1: yield: 75%. 1H NMR (500 MHz, DMSO) δ 10.92 (d, J = 46.8
Hz, 1H), 7.89 (d, J = 99.0 Hz, 2H), 4.59 (d, J = 7.3 Hz, 2H), 4.40 (d,
J = 13.3 Hz, 2H), 4.20 (d, J = 4.5 Hz, 4H), 2.14 (t, J = 11.3 Hz, 1H),
1.05 (dd, J = 12.6, 7.5 Hz, 3H).
L2: yield: 80%. 1H NMR (500 MHz, DMSO) δ 10.04 (d, J = 28.1
Hz, 1H), 4.58 (d, J = 23.3 Hz, 2H), 4.35 (d, J = 11.7 Hz, 2H), 4.17
(d, J = 8.1 Hz, 4H), 2.58−2.54 (m, 1H), 2.25 (dd, J = 14.8, 7.3 Hz,
1H), 2.12 (d, J = 10.2 Hz, 3H), 1.05 (t, J = 7.5 Hz, 3H).
MATERIALS AND INSTRUMENTATION
■
Unless otherwise noted, all manipulations were performed using
standard Schlenk tube techniques under nitrogen atmosphere. The
reagents ferrocenealdehyde (≥98.0%), acetylferrocene (≥97.0%),
hydrazine hydrate (≥50%), Ph3SnCl (≥60%), and propionic acid
hydrazine were purchased from Sigma-Aldrich. For the biological
experiments, Hoechst 33342(apoptosis and epigenetice company), 3-
methyladenine (apoptosis and epigenetice company), cycloheximide,
leupeptin, necrostatin-1, carbonyl cyanide m-chlorophenylhydrazone
(CCCP), Z-VAD-FMK, and cleaved caspase-3 were purchased from
Apoptosis and Epigenetics Company. MTDR (Life Technologies),
LTDR (Life Technologies), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Sigma-Aldrich), Annexin V-FITC
Apoptosis Detection Kit (Sigma-Aldrich), JC-1 (Sigma-Aldrich),
and PI (Sigma-Aldrich) are all used as received. EDTA (30 mM) was
purchased from TaKaRa Biotechnology (Dalian, China). DMEM
medium, fetal bovine serum, penicillin/streptomycin mixture, trypsin/
EDTA, and PBS were purchased from Sangon Biotech. Testing
compounds were dissolved in DMSO and diluted with the tissue
culture medium before use.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b03305.
Experimental section, design strategy, stability moni-
tored by UV−vis spectra, 1H NMR spectra, electrostatic
potential models, flow cytometry results, confocal
microscopy images, cell cycle arrest results, crystallo-
graphic data, selected bond lengths and angles, growth
inhibition, apoptosis, and caspase 3 activity results,
MMP data, ROS induction, cell cycle analysis (PDF)
Accession Codes
CCDC 1843945 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
SYNTHETIC PROCEDURES
The synthesis of new ferrocenyl−organotin compounds is shown in
Scheme 1.
■
Synthesis of β-Methoxycarbonyl Ethyl Triphenyltin (S1). S1
was prepared according to literature methods.28
Synthesis of β-Hydrazinocarbonyl Ethyl Triphenyltin (S2).
S1 (2.0 g, 4.5 mmol) and methanol (30 mL) were added into a 250
mL three-necked bottle under N2, followed by stirring and heating to
ebullience for 20 min. Hydrazine hydrate (50%, 30 mL) was added
dropwise to above solution at 80 °C. The reaction was not stopped
until S1 was consumed completely (monitored by thin-layer
chromatography). Crude product was isolated by filtration and
recrystallized from 60 mL of mixtures of chloroform and petroleum
ether (v/v = 1:1) to obtain a colorless acicular crystal. Yield: 80.1%.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: chemlxc@163.com (X.L.).
*E-mail: liuzheqd@163.com (Z.L.).
ORCID
Xicheng Liu: 0000-0002-5932-7206
Zhe Liu: 0000-0001-5796-4335
1
Mp, 144.2−144.5 °C. H NMR (500 MHz, CDCl3) δ 7.56 (dt, J =
G
DOI: 10.1021/acs.inorgchem.8b03305
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the University Research Development Program of
Shandong Province (J18KA082), the National Natural Science
Foundation of China (Grant No. 21671118) and the Taishan
Scholars Program for support.
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